Thesis Submitted for the Degree
of
Candidatus Scientiarum
Electron Paramagnetic
Resonance Studies of the
Mixed Valence DiironOxygen Cluster in the
Mouse Ribonucleotide
Reductase R2 Protein
by
Åsmund Kjendseth Røhr
Department of Biochemistry
The Faculty of
Mathematics and Natural Sciences
University of Oslo
Oslo 2001
1
Electron Paramagnetic Resonance Studies of the Mixed
Valence Diiron-Oxygen Cluster in the Mouse Ribonucleotide
Reductase R2 Protein
III
Acknowledgments
The work presented in this thesis has been carried out at the Department of
Biochemistry, University of Oslo under supervision of Professor K. Kristoffer
Andersson.
Professor K. Kristoffer Andersson has provided valuable supervision and given me
the opportunity to learn about the interesting field of metalloprotein research. He has
also provided financial support to me when visiting conferences. I am very grateful
for all his care during my last years at the University of Oslo.
I will also thank my co-supervisor Dr. Peter Paul Schmidt for all help and
encouragement. He trained me in EPR spectroscopy and the basic lab work.
Dr. Morten Sørlie and Cand. Scient. Kari Røren Strand have read through this thesis. I
want to thank them for correcting my language and for helpful discussions regarding
the scientific presentation of my work.
Dr. Joshua Telser has kindly provided computer software for analysis of EPR spectra,
and valuable advise on how to make it work. Professor Einar Sagstuen helped me to
compile and modify the source code from Dr. Telser. I want to thank them for their
valuable help.
Thanks to far, mor, Torolf, and Kristin for all support and encouragement!
Oslo, 20.05.2001
Åsmund Kjendseth Røhr
V
Table of Contents
1 Abstract ___________________________________________________________ 1
2 Introduction________________________________________________________ 3
2.1 Preface ______________________________________________________________ 3
2.2 Ribonucleotide Reductase ______________________________________________ 4
2.2.1 General Introduction ________________________________________________________ 4
2.2.2 The Diiron-Oxygen Cluster and Tyrosyl Radical in RNR R2 ________________________ 6
2.3 Methane Monooxygenase _______________________________________________ 9
2.3.1 General Introduction ________________________________________________________ 9
2.3.2 The Diiron-Oxygen Cluster in MMOH ________________________________________ 10
2.4 Uteroferrin__________________________________________________________ 12
2.4.1 General Introduction _______________________________________________________ 12
2.4.2 The Diiron-Oxygen Cluster in Uteroferrin ______________________________________ 12
2.5 Spectroscopical Properties of Diiron-Oxygen Clusters ______________________ 14
2.5.1 General Spectroscopic Properties of Different Oxidation States _____________________ 14
2.5.2 Application of EPR to Diiron-Oxygen Clusters __________________________________ 15
2.6 Aims for the Thesis ___________________________________________________ 17
3 Methods __________________________________________________________ 19
3.1 Expression of the Mouse R2 Gene in E.coli _______________________________ 19
3.2 Purification of Mouse R2 Protein _______________________________________ 20
3.2.1 Lysis of Bacteria __________________________________________________________ 20
3.2.2 Precipitation of DNA ______________________________________________________ 20
3.2.2 Precipitation of R2 Protein with Ammonium Sulfate ______________________________ 21
3.2.3 Gel Filtration Chromatography_______________________________________________ 21
3.2.4 Anion Exchange Chromatography ____________________________________________ 22
3.2.5 Sodium Dodecyl Sulfate Polyacrylamide Electrophoresis __________________________ 22
3.2.6 Ultra Filtration ___________________________________________________________ 23
3.3 Protein Quantification ________________________________________________ 24
3.3.1 Quantification Using UV/vis Spectrophotometry _________________________________ 24
3.3.2 Bio-Rad Protein Assay _____________________________________________________ 24
3.4 Buffer Exchange and Ultra Filtration ____________________________________ 25
3.5 Reconstitution of the Diiron-Oxygen Cluster and the Tyrosyl Radical _________ 25
3.6 Quantification of Dithionite ____________________________________________ 26
3.7 Anaerobic Reduction of Phenazine Methosulfate __________________________ 26
3.8 Reduction of the Diiron-Oxygen Cluster and the Tyrosyl Radical ____________ 27
3.9 Electron Paramagnetic Resonance Spectroscopy __________________________ 29
3.9.1 Introduction to EPR Theory _________________________________________________ 29
3.9.2 The Electronic Zeeman Effect _______________________________________________ 30
3.9.3 Zero Field Splitting ________________________________________________________ 33
3.9.4 Exchange Coupling________________________________________________________ 34
3.9.5 The Spin Hamiltonian for a Dinuclear Coupled Metal Cluster_______________________ 35
3.9.6 Relaxation of a Paramagnetic System _________________________________________ 35
3.9.7 EPR Instrument Parameters Used in the Experiments _____________________________ 37
3.9.8 EPR Sample Preparation ___________________________________________________ 37
3.9.9 Quantification of Spin in an EPR Sample ______________________________________ 37
3.10 Circular Dichroism Spectroscopy ______________________________________ 38
VI
4 Results and Analysis ________________________________________________ 39
4.1 Protein Purification __________________________________________________ 39
4.2 Reconstitution of Mouse RNR-R2 _______________________________________ 39
4.3 Redox Studies of Phenazine Methosulfate ________________________________ 40
4.3.1 Purpose of the Experiments _________________________________________________ 40
4.3.2 The Equilibrium between Different Redox Forms of PMS _________________________ 41
4.4 Reduction of the Tyrosyl Radical and the Diiron-Oxygen Cluster ____________ 42
4.4.1 Purpose of the Experiments _________________________________________________ 42
4.4.3 Estimation of the Midpoint Potential E m’ of the Diiron-Oxygen Cluster _______________ 44
4.5 Interactions between Alcohols and the Diiron – Oxygen Cluster ______________ 48
4.5.1 Purpose of the Experiments _________________________________________________ 48
4.5.2 Affinity of Various Primary Alcohols to the Mixed Valence Cluster _________________ 48
4.5.3 Estimation of Binding Constants for Methanol and Ethanol with Mouse R2 ____________ 50
4.5.4 Effect of Isotope Labeled Alcohols ___________________________________________ 52
4.5.5 Microwave Powersaturation Behavior of the Novel EPR Signals ____________________ 52
4.6 Theoretical Studies of the Mouse R2 Mixed Valence Cluster _________________ 55
4.6.1 Purpose of the Theoretical Studies ____________________________________________ 55
4.6.2 Simulation of Experimental EPR Spectra _______________________________________ 56
4.6.3 Ligand Field Calculations ___________________________________________________ 60
4.6.3 Summary of Results From Theoretical Calculations ______________________________ 63
4.7 CD and Light Absorption Studies of the Mouse R2 Diferric Cluster __________ 64
4.7.1 Purpose of the Experiments _________________________________________________ 64
4.7.2 Reconstituted Mouse R2 in the Presence of Methanol _____________________________ 64
5 Discussion ________________________________________________________ 67
5.1 Introduction_________________________________________________________ 67
5.2 Tyrosyl Radical Content in Reconstituted Mouse R2 _______________________ 67
5.3 Redox Chemistry of PMS and Mouse R2 _________________________________ 67
5.4 Small Alcohols might Bind to the Mouse R2 Mixed Valence Cluster __________ 69
5.4.1 Relevance of Results and Further Experiments __________________________ 75
6 Appendix _________________________________________________________ 77
6.1 Materials ___________________________________________________________ 77
6.2 The Culture Medium and Buffers _______________________________________ 79
6.3 Input Parameters for the Program ddpowjea _____________________________ 82
Terms and Abbreviations ______________________________________________ 83
References _________________________________________________________ 85
VII
VIII
1 Abstract
The enzyme ribonucleotide reductase (RNR), important for all life, catalyses the
reduction of ribonucleotides to their corresponding deoxyribonucleotides. The R2
homodimer of the enzyme complex contains an -oxo bridged diiron-oxygen cluster
and a tyrosyl radical that are essential for the enzymatic reaction. Similar diironoxygen clusters exist in the enzymes methane monooxygenase hydroxylase (MMOH)
and uteroferrin. We have studied the mixed valence Fe(II)-Fe(III) oxidation state of
the diiron-oxygen cluster in mouse R2 by electron paramagnetic spectroscopy (EPR).
From the results of introductory redox studies we estimate a midpoint potential (Em’)
to be between 52 mV and 62 mV (versus the SHE) for the reduction of the diironoxygen cluster from the Fe(III)-Fe(III) oxidation state to Fe(II)-Fe(II). These
experiments resulted in a maximum yield of 0.56 mixed valence clusters per R2
dimer.
A novel interaction between alcohols and the mouse R2 mixed valence cluster was
characterized. Results from titration experiments indicate binding constants of Kb,
methanol
= 0.24  0.02 M and Kb, ethanol = 0.60  0.03 M. These constants were estimated
from the observed perturbations of experimental EPR spectra induced by methanol
and ethanol, respectively. Our hypothesis that alcohol interact with the mixed valence
cluster is supported by theoretical calculations. These calculations indicate that the
ligand field of the ferrous iron in the mixed valence cluster is modulated by the
interaction of methanol and ethanol.
Altogether, our results suggest that the mixed valence diiron-oxygen cluster in mouse
R2 and MMOH have similar properties. The Em’ value determined for the reduction
of the diiron-oxygen cluster in MMOH (OB3b)1 is close to our estimate for the mouse
R2. Results form theoretical calculations performed at the methanol-mixed valence
cluster in MMOH2 and mouse R2 are comparable. Thus we suggest that mouse R2 is
an adequate starting point when engineering a MMOH like enzyme from R2.
1
2 Introduction
2.1 Preface
In this thesis, all experimental work has been performed using the R2 subunit of
mouse ribonucleotide reductase (RNR). The research has been focused around the
properties of the diiron-oxygen cluster in this protein and not the overall function of
the enzymatically active R1-R2 complex. Thus, it is natural to give a general
overview of several proteins that contain diiron-oxygen clusters similar to the one
found in mouse R2. The proteins to be introduced are listed in Table 2.1 with their
catalytic properties. A detailed presentation of the mixed valence Fe(II)-Fe(III)
oxidation states of the metal clusters in these proteins are given at the end of this
chapter. The discussion of the results presented in this thesis will be related to the
properties of the different diiron-oxygen clusters presented here.
Table 2.1 Diiron-oxygen proteins have different functions a
Enzyme
Reaction type
Catalytic reaction
Ribonucleotide reductase R2
1e- oxidation
Fe(II)Fe(II)  TyrOFe(III)Fe(III)  Tyr
Methane monooxygenase b
hydroxylation
Fe(II)Fe(II)  CH4 OFe(III)Fe(III)  CH3OH
Uteroferrin
hydrolysis of
phosphate ester
Fe(II)Fe(III)  ROHPO3 HOFe(II)Fe(III)  H3PO4
2
2
2
a
Information obtained from Solomon et al.3 b Methane hydroxylation is catalyzed by the hydroxylase
component of the methane monooxygenase complex.
Other proteins such as stearoyl-acyl carrier protein 9 desaturase and the invertebrate
oxygen transporting protein hemerythrin also contain diiron-oxygen clusters
comparable to the one in R2. Solomon et al.3 have recently published an excellent
review regarding the geometric and electronic structure of the diiron-oxygen clusters
in these proteins.
3
Chapter 2
Introduction
2.2 Ribonucleotide Reductase
2.2.1 General Introduction
Class I ribonucleotide reductases are found in eukaryotic and prokaryotic organisms,
and some viruses that have nucleic acids coding for viral RNR. Other classes of RNR
also exist. These are described in the literature4,5 and will not be discussed here. The
holoenzyme of RNR catalyses the reduction of ribonucleotides to
deoxyribonucleotides (Scheme 2.1).
O
HO
P
O
O
P
O
BASE
O
HO
P
O
O
P
BASE
O
O
OH
O
OH
OH
OH
OH
OH
OH
Scheme 2.1
The catalytic active form of class I RNR is composed of two homodimers named R1
(2 x ~85 kD) and R2 (2 x ~43 kD). From X-ray crystallography studies, it has been
determined that the R1 monomer contains / barrel, -helical and + domains. The
active and regulatory sites are also located in this subunit.6,7 The R2 monomer has
mainly an -helical bundle tertiary structure. It can bind two ferrous iron atoms that
can react with dioxygen to form a diferric -oxo bridged diiron-oxygen cluster and a
tyrosyl radical. This radical is essential for enzymatic activity.8-10 A proposed
structure of the E.coli R1-R2 holoenzyme is visualized in Figure 2.1.5
A radical based reaction mechanism has been proposed for the ribonucleotide
reduction.12 According to this suggestion, a radical should be either on the 3’ or
2’carbon in the substrate, or on the catalytic active cysteines in the R1 subunit during
catalysis. This hypothesis has been supported by studies where cytidine analogs were
used to trap a radical at the R1 active site.13-15
An hydrogen atom or electron/H+ transfer16 pathway from the tyrosyl radical located
in the R2 subunit to the active site at R1 has been proposed in order to explain how
the radical can be translocated the distance of ~35 Å separating the tyrosyl radical and
the active site during catalysis.5
4
Chapter 2
Introduction
The hydrogen bonded amino acid sidechains suggested to be involved in the radical
transfer have been altered by site directed mutagenesis in order to verify that
Active site
R1
Regulatory
site
R2
Diiron-oxygen
cluster and Tyr
Figure 2.1 A proposed model of the E.coli R1-R2 holoenzyme. The model is based
on an illustration published by B.-M. Sjöberg.{4} -helixes are colored yellow and
blue in the R1 and R2 homodimers, respectively. -sheets are colored white in both
homodimers. Red spacefill representations indicate important sites. The model was
created from the PDB files 1RIB and 2R1R with the program MolMol. 11
hypothesis. Results from experiments performed with both E.coli and mouse R2
confirmed that the proposed amino acids were essential for catalysis.5,17 The
importance of an intact hydrogen bonded pathway in R2 has also been demonstrated
for the reaction between the ferrous iron atoms and a dioxygen molecule in the R2
subunit.17,18 It should also be mentioned that the proposed radical transfer pathway
includes one of the iron atoms in the diiron-oxygen cluster. Thus, a mixed valence
Fe(II)-Fe(III) oxidation state of the diiron-oxygen cluster might has a physiological
relevance.
5
Chapter 2
Introduction
Detailed information regarding the radical and the diiron-oxygen clusters in R2
proteins will be of great value for medical research when inhibitors specific for the
radical and the metal cluster are to be constructed. Inhibition of RNR results in
depletion of the deoxyribonucleotide pools in the cells, thus arresting cell division.
Three common approaches for RNR inhibition are; inhibitors that binds to the active
sites at the R1 homodimer,19 peptides that resembles the R2 interface to the R1
homodimer and prohibit the indispensable R1 – R2 interaction,20 and third; inhibitors
that quench the radical and the metal center in the R2 homodimer that is essential for
catalysis.21,22 An example of such an inhibitor is hydroxyurea that eliminates both the
redox active tyrosyl radical and the diiron-oxygen cluster in mammalian R2.21,22
When the E.coli RNR enzyme is used as a model for the mouse RNR in this thesis,
the amino acid numbering is given as e.g. Y122(177), which point towards tyrosine
122 in E.coli and tyrosine 177 in mouse.
2.2.2 The Diiron-Oxygen Cluster and Tyrosyl Radical in RNR R2
In the active holo-RNR enzyme complex, the function of the R2 subunit is to supply a
radical to the active site at R1 during catalysis. R2 also provide a protected
environment for the radical when no substrate is bound at R1.
The most extensively studied class I RNR R2 protein is the one from E.coli. An
approach for the in vitro ferrous iron (Fe2+) dioxygen reaction in E.coli R2 was
published by Brown et al.23 in 1969. Results from early Mössbauer and light
absorption experiments showed that a diferric (Fe3+-Fe3+) antiferromagnetically
coupled cluster was formed in addition to the tyrosyl radical.24 Since then, numerous
studies have been focused at the iron-oxygen reaction, and its intermediates, that ends
with the generation of a tyrosyl radical. A detailed illustration of the proposed steps in
the in vitro reconstitution reaction is illustrated in Scheme 2.2 mainly adopted from
Andersson et al.25
6
Chapter 2
Introduction
It is necessary to explain the individual steps in
Scheme 2.2, and E.coli R2 amino acid
numbering is used throughout. At the starting
point D84, E238, E204, H241, E115, and H118
bind two ferrous iron atoms. The low
Diferrous
coordination numbers of the irons allow
binding of dioxygen to one of the irons.26
Crystallographic studies have shown that azide
(N3-) binds in a specific fashion to the reduced
diiron-oxygen cluster, and the O2 bound
complex have been modeled from these
O2 bound
results.25 During dioxygen cleavage, a peroxo
species have been observed in the mutant D84E
R2 protein.27 This intermediate, called
compound P, is proposed in the native R2 ironoxygen reaction as well. In the next step, the
peroxo bridge is reduced to a water molecule
Peroxo
and a -oxo bridge by two electrons. One
electron is provided from one of the ferric irons
of the cluster and the second electron is donated
from an external source that usually is Fe2+ in
the in vitro reconstitution reaction. This results
in a -oxo bridged Fe(IV)-Fe(III) mixed
valence cluster with a S = ½ ground state that
Compound X
has been observed by electron paramagnetic
resonance (EPR) and Mössbauer
spectroscopy.28,29 This high-valence diironoxygen cluster, termed compound X, is not
stable, and is assumed to abstract an electron
from Y122(177) to form the unprotonated
Diferric + Tyr
tyrosyl radical.3
Scheme 2.2
7
Chapter 2
Introduction
When the three dimensional structure
of E.coli R2 was published in 1990 by
Nordlund et al.,8 it became clear that
the radical containing Y122(177) was
located ~5 Å away from the diironoxygen cluster. The crystal structure of
the E.coli R2 monomer with a diferric
cluster is shown in Figure 2.2. As in
several other diiron-oxygen proteins,
carboxyls and histidines inside a fourhelix bundle ligate the metal cluster. In
its diferric form, both irons are sixcoordinated (Figure 2.3). It is not
agreed upon whether the reduced form
have two four-coordinated irons26 or
one four and one five-coordinated
Figure 2.2 Structure of E.coli R2 (monomer)
containing a diferric cluster. Y122(177) is purple
while iron ligating sidechains are red. The
illustration was created from the PDB file 1RIB
with the program MolMol.11
iron.30 From crystallographic studies it
have been shown that only the
positions of the amino acid sidechains
involved in coordination of the diironoxygen cluster are changed for the apo,
the diferrous, and the diferric forms of
E.coli R2.31 In 1996, the crystal
structure of mouse R2 was solved by
Kauppi et al.32 Unfortunately only one
iron was observed to be bound inside
the protein, and the crystal was grown
at the unphysiological pH 4.7. Hence,
Figure 2.3 The diferric -oxo bridged diironoxygen cluster from E.coli R2. Each iron is
coordinated by one His and one water molecule. All
other ligands are carboxyls. The illustration was
created from the PDB file 1RIB with the program
MolMol.11
there is no atom coordinates for the mouse R2 diiron-oxygen cluster available.
However, it was observed that the pocket where the diiron-oxygen cluster is located is
solvent exposed. This is not the case for E.coli R2 where the diiron-oxygen cluster is
inaccessible. The observations are supported by the fact that the mouse R2 tyrosyl
radical is more sensitive to scavengers than the E.coli R2 radical.33,34
8
Chapter 2
Introduction
Other experiments have also confirmed differences between mouse and E.coli R2.
First, results from high field EPR experiments have suggested the presence of a
hydrogen bond to the unprotonated Y177 sidechain in mouse R2 that is not present in
E.coli R2.35 Second, the two irons in mouse R2 obviously have different redox
potentials because a mixed valence Fe(II)-Fe(III) state can be obtained by chemical
reduction of the Fe(III)-Fe(III) state.36,37 In native E.coli R2, the two irons are reduced
simultaneously at a lower redox potential than suggested for the diiron-oxygen cluster
in mouse R2.38 These differences suggest that even though the iron coordinating
amino acids are highly conserved, slight variations of the structures give the diironoxygen clusters in mouse and E.coli R2 individual chemical and physical properties.
2.3 Methane Monooxygenase
2.3.1 General Introduction
Anaerobic methanogenic bacteria living in oceans, lakes, and wet soils produce large
amounts of methane. In the aerobic environment above the methanogenic bacteria
habitat, methanotropic bacteria metabolize methane into methanol. Methanol is the
only carbon source for these methanotropic bacteria, and it is converted into biomass
and CO2.39
In this chapter, only the soluble forms of the enzyme methane monooxygenase
(MMO) from Methylococcus capsulatus (Bath) and Methylosinus trichosporium
(OB3b) will be discussed. The reaction catalyzed by methane monooxygenase is
shown in Scheme 2.3.
CH 4  O 2  NAD(P)H  H   CH 3 OH  NAD(P)   H 2 O
Scheme 2.3
In brief, three protein components are required for catalytic activity:39
1. Hydroxylase (MMOH), a hetrotrimer  (~245 kD) with a quaternary structure
()2 and a diiron-oxygen cluster active site located in subunit .
9
Chapter 2
Introduction
2. Reductase (MMOR), a monomeric protein (~38 kD) with a Fe2S2 cluster that
transfer electrons from NAD(P)H to the MMOH diiron-oxygen cluster.
3. Component B, a regulatory monomeric protein (~15 kD) able of inducing
conformational changes in the hydroxylase.
2.3.2 The Diiron-Oxygen Cluster in MMOH
In 1986 it was suggested that MMOH
(Bath) probably contained a diironoxygen cluster.40 Mössbauer studies of
MMOH (OB3b) suggested that the
resting state of the enzyme had a
hydroxide-bridged diferric cluster.2
Pulsed EPR studies also revealed that
the one electron reduced mixed
valence Fe(II)-Fe(III) form also had a
hydroxide bridge.41 The ligation of the
mixed valence clusters in both OB3b42
and Bath43 MMOH was further
investigated by spectroscopy, and
histidine coordination of the diironoxygen was suggested for both types
Figure 2.4 Structure of the -subunit of MMOH.
The diferric cluster and its protein ligands are
orange and red, respectively. The illustration was
created from the PDB file 1MTY with the
program MolMol.11
of MMOH. The first crystal structures
of Bath44 and OB3b45 MMOH were
determined in 1993 and 1997,
respectively. Both proteins had high
structural similarity and a diironoxygen cluster in the -subunit. It was
also concluded that either two
hydroxides, or one hydroxide and one
water molecule, bridged the two
irons.45
Figure 2.5 The diferric cluster in MMOH is very
similar to the one in E.coli R2. The illustration
was created from the PDB file 1MTY with the
program MolMol.11
10
Chapter 2
Introduction
The structure of the MMOH (Bath)  subunit determined at 1.7 Å resolution
published in 1997 by Rosenzweig et al.46 is shown in Figure 2.4. The diferric clusters
of MMOH (illustrated in Figure 2.5) and E.coli R2 (Figure 2.3) show a high degree of
structural similarity. Instead of a -oxo bridge as observed in E.coli R2, a water and a
hydroxide have bridging positions in the diferric MMOH. In E.coli R2, one of the
carboxyl ligands is an aspartate (D84). This amino acid is a glutamate in MMOH.
Recently, new structures of MMOH (Bath) have been determined with the diironoxygen cluster in oxidized Fe(III)-Fe(III), mixed valence Fe(II)-Fe(III) and reduced
Fe(II)-Fe(II) forms, revealing that the coordination numbers of the irons depend on
the oxidation state.47
The catalytic cycle of MMO results in
hydroxylation of the substrate. As in class I
RNR-R2, a high valent diiron-oxygen cluster
carry out the substrate oxidation.
Characterized key intermediates in the
MMOH iron-oxygen reaction are illustrated
in Scheme 2.3, which is taken from Shu et
al.48 Several similarities are found for the
diferrous R2 and MMOH dioxygen
activation process. Intermediate P, a
peroxide species characterized in MMOH,49
Scheme 2.3
resembles compound P in R2. While
compound X in R2 has a Fe(III)-Fe(IV) oxidation state, the relative in MMOH,
termed intermediate Q, has been determined to be a Fe(IV)-Fe(IV) species.48
Cryoreduction of intermediate Q by -radiation results in a Fe(III)-Fe(IV) iron-oxygen
cluster that resembles compound X.50 By considering these data, it is apparent that in
addition to very similar iron coordination environments in R2 and MMOH, and that
the two proteins activate dioxygen in an analogous fashion.
It has been observed that the product release from diferric MMOH is the rate limiting
step during catalysis.51 The alcoholdiferric cluster complex termed compound T (T
for terminal adduct) has been studied by X-ray crystallography, and it was shown that
11
Chapter 2
Introduction
methanol was bound to both irons whereas ethanol was coordinated to a single iron.52
Radiolytic reduction of the diferric cluster by -irradiation at 77 K in presence and
absence of methanol followed by EPR studies also indicated that methanol interacted
with the diferric cluster.53 Electron nuclear double resonance (ENDOR) analysis of
the mixed valence Fe(II)-Fe(III) cluster in the presence of methanol, demonstrated
that the alcohol was coordinated to the ferrous iron.54 A simple ligand field model was
used to explain the methanol-ferrous iron interaction on the basis of a combined
Mössbauer/ EPR study of the MMOH (OB3b) mixed valence cluster.2 This ligand
field model has also been used to explain a novel interaction between alcohols and the
mouse R2 mixed valence cluster in this thesis.
2.4 Uteroferrin
2.4.1 General Introduction
Uteroferrin (~35 kD) is a purple acid phosphatase that can be isolated from pig
allantoic fluid. Purple acid phosphatases are a highly homologous group of enzymes
that catalyze nonspecific cleavage of phosphoester bonds as illustrated in Scheme 2.4,
and are found in plants, fungi, and animals.55
H 2O
R  OHPO 3 
 R  OH  H 2 PO 4
Scheme 2.4
The mammalian type has been suggested to be involved in osteoporosis56 and
immunologic response to pathogens.57
2.4.2 The Diiron-Oxygen Cluster in Uteroferrin
In 1983, the first evidence for a dinuclear iron-cluster in uteroferrin was obtained
from EPR spectroscopy.58 Subsequent Mössbauer spectroscopy studies verified the
presence of an antiferromagnetically coupled diiron cluster.59 In contrast to R2 and
MMOH, the active form of uteroferrin contain a mixed valence Fe(II)-Fe(III) diiron-
12
Chapter 2
Introduction
oxygen cluster, and dioxygen
activation is not a part of the catalytic
mechanism.55 It was suggested that the
antiferromagnetic coupling was weak
from results obtained from magnetic
circular dichroism (MCD)60 and
Mössbauer61 experiments. These
indicated a bridging hydroxide in the
active Fe(II)-Fe(III) cluster.
A plausible reaction mechanism for the
Scheme 2.5
hydrolysis of phosphate suggested by
Yang et al.60 is depicted in Scheme 2.5. The crystal structure of uteroferrin was solved
in 1999 and is illustrated in Figure 2.6.62 It shows a different tertiary organization of
the polypeptide chain compared to R2 and MMOH.
While R2 and MMOH have mainly helical structure, uteroferrin has a -sandwich
structure that is common to purple acid phosphatases. The diiron-oxygen clusters in
R2 and MMOH are buried in four helix bundles while the active site in uteroferrin is
located at the surface of the protein. The diiron-oxygen cluster in uteroferrin is
illustrated in Figure 2.7, and the ligand arrangement is different from the one in R2.
An additional histidine coordinates to the uteroferrin diiron-oxygen cluster, and a
Figure 2.6 The crystal structure of uteroferrin
determined at 1.55 Å resolution. The -sandwich
structure distinguishes this protein from the mainly
helical R2 and MMOH proteins. The illustration
was created from the PDB file 1UTE with the
program MolMol.11
Figure 2.7 The diferric oxygen-bridged cluster in
uteroferrin have quit different ligation than the R2
and MMOH cluster. The illustration was created
from the PDB file 1UTE with the program MolMol. 11
13
Chapter 2
Introduction
tyrosine and an asparagine have replaced two carboxyl ligands. The purple color of
concentrated enzyme solutions originates from charge transfer transitions between the
ferric iron and its tyrosine ligand.55
2.5 Spectroscopical Properties of Diiron-Oxygen Clusters
2.5.1 General Spectroscopic Properties of Different Oxidation States
Characterized binuclear iron clusters in proteins mainly have either oxygen or sulfur
based bridges. The proteins introduced in the previous sections contain binuclear iron
clusters that have oxygen, hydroxide, water, and carboxyl bridges. The properties of
these clusters are generally not well understood compared to Fe2S2 clusters.
The irons in the diiron-oxygen clusters are generally high spin. This means that the
electrons are distributed in the three non-bonding and the two antibonding d-orbitals.
This results in S = 5/2 for high spin Fe(III) and S = 2 for high spin Fe(II). When two
such irons have one or more bridging ligand(s), the spins are either ferromagnetically
or antiferromagnetically coupled. Such couplings implicate that the two spin vectors
of each iron are added or subtracted, and that the total spin is the resultant vector (see
Chapter 3.9.4 for a detailed explanation). Possible spin combinations in a diiron
cluster are listed in Table 2.2.
Table 2.2 Spin states in coupled high spin diiron clusters
a
Oxidation state
Individual spins
Stotal (antiferromagnetic)a
Stotal (ferromagnetic)a
Fe(III)-Fe(III)
5/2……5/2
0
5
Fe(II)-Fe(III)
2…...…5/2
½
9/2
Fe(II)-Fe(II)
2…….…2
0
4
Only ground states are given here.
For mouse R2,36,37,63 MMOH,40,64 and uteroferrin,58,65 the diiron-oxygen clusters in
Fe(III)-Fe(III) and Fe(II)-Fe(III) oxidation states are antiferromagnetically coupled.
The Fe(III)-Fe(III) cluster of E.coli R2 is also antiferromagnetically coupled.63
14
Chapter 2
Introduction
However, the Fe(II)-Fe(III) form of the E.coli R2 cluster obtained by radiolytic
reduction by -irradiation at 77 K can be ferromagnetically coupled.66
The magnitude of the magnetic coupling between the irons is given by the exchange
constant J (see Chapter 3.9.4). Antiferromagnetic and ferromagnetic coupling
constants have values J < 0 and J > 0, respectively. Coupling constants for diironoxygen clusters in different oxidation states are given in Table 2.3. A value of J larger
than approximately -35 cm-1 and lower than about -5 cm-1 is considered as a signature
of a protonated oxo-bridge.67
Table 2.3 Exchange coupling constants for diiron-oxygen clusters in proteins
J (cm-1)
Protein
Fe(III)-Fe(III)
Fe(II)-Fe(III)
Fe(II)-Fe(II)
Mouse R237,63
-77
-30 < J < -10a
J > 0b
E.coli R230,63,66
-92
6
~ -0.5
MMOH2,68
-7.5
-30
~ 0.5
-300 < J < -80
-16 < J < -5
-
58,60,61,65
Uteroferrin
a
J has not been properly determined. This interval is assumed on the basis of the work presented in this
thesis.
b
Kari Røren Strand, personal communication.
The values presented in Table 2.3, and other parameters describing the electronic
structures of these diiron-oxygen clusters, have been estimated by spectroscopical
analysis. Mössbauer (57Fe -radiation absorption) and magnetic circular dichroism
(MCD) are excellent techniques for studying diiron-oxygen clusters in the redoxstates shown in Table 2.2. Electron paramagnetic resonance (EPR is a suitable
technique when studying half-integer spin systems. Complimentary information
regarding the electronic structure of a spin system can be obtained by using the three
techniques mentioned above.
2.5.2 Application of EPR to Diiron-Oxygen Clusters
The diiron-oxygen clusters in R2, MMOH and uteroferrin all have one or more
paramagnetic EPR active oxidation states. Information is especially obtained from the
15
Chapter 2
Introduction
mixed valence Fe(II)-Fe(III) oxidation
2.004
these species are briefly discussed
below.
Low temperature EPR spectra
recorded for the mouse R2 are
EPR FIRST DERIVATIVE
states, and EPR spectra recorded for
A
1.92
gobs= 12.5
1.73
B
1.60
illustrated in Figure 2.8. Only the
1000
narrow S = ½ tyrosyl radical signal,
2000
3000
4000
Field (G)
which is expanded in Figure 2.8, is
detected for the Fe(III)-Fe(III) active
form (spectrum A). When reduced, the
tyrosyl radical signal at geff = 2
disappears, and two new signals arise;
1) the mixed valence Fe(II)-Fe(III)
form that has a S = ½ ground state, is
Figure 2.8 EPR spectra of mouse R2 in different
oxidation states. Spectrum A; active R2 where only
the tyrosyl radical is detected. Spectrum B; reduced
forms of mouse R2. The mixed valence form and
the diferrous form have g- values 12.5 and (1.92,
1.73, 1.60), respectively. A narrow signal at geff = 2
in spectrum B originates from the electron mediator
that was used to reduce the active form. Conditions:
(A); 15 K, 100W. (B); 4 K, 20 mW. Microwave
frequency 9.6 GHz used for both spectra.
identified by the rhombic EPR signal
with effective g-values 1.92, 1.73 and
A
1.60. 2) an integer S = 4 signal
originating from ferrous iron is
observed at geff = 12.5.
B
In the proceeding chapters, the mixed
valence EPR spectra obtained with the
mouse R2 will be compared to spectra
that previously have been obtained
C
with MMOH and uteroferrin. Thus,
these spectra have been depicted in a
shared illustration to make
comparisons easier. In Figure 2.9, the
effective g-values for the various
Figure 2.9 Mixed valence Fe(II)-Fe(III) X-band
EPR spectra of A; MMOH, B; mouse R2 and C;
uteroferrin. All spectra have been recorded at
low temperature. See references in text.
mixed valence species are; MMOH
(Bath)42 (1.94, 1.86, 1.76), mouse R2
16
Chapter 2
Introduction
(1.92, 1.73, 1.60), and uteroferrin58 (1.93, 1,74, 1.59). The large variations in the gvalues are related to the structure of the clusters. In brief, a combination of the
exchange coupling occurring via the Fe(II)-Fe(III) bridge and the ligand field induced
by coordinating atoms strongly affects the broadness of the signal and the positions of
the peaks. The theory concerning EPR and ligand field are described and discussed in
Chapter 3.9 and 4.6, respectively.
2.6 Aims for the Thesis

Conduct introductory studies of the redox properties of the diiron-oxygen cluster
in mouse R2. The main purposes of these studies was to:
1. Achieve a high yield of the mixed valence oxidation state of the diiron-oxygen
cluster.
2. Estimate the midpoint potential for the reduction of the Fe(III)-Fe(III) cluster
to the Fe(II)-Fe(II) oxidation state.

Characterize the nature of a novel interaction between alcohol and the mouse R2
mixed valence cluster.
17
3 Methods
All materials utilized are listed in Chapter 6.1. The culture medium and buffer recipes
are described in Chapter 6.2. Abbreviations for these solutions are used throughout
the text.
3.1 Expression of the Mouse R2 Gene in E.coli
The E.coli bacteria strain BL21 (DE3) expressing the recombinant mouse RNR-R2
gene69 was kindly provided by Lars Thelander, University of Umeå.
Principle:
The mouse R2 gene is expressed in a T7 RNA polymerase expression system being
activated by Isopropyl--D-Thiogalactopyranoside (IPTG).70,71
Procedure:
The following procedure have been described by Mann et al..69 As a rule the bacteria
were grown in LB culture medium containing 50 g/mL carbenicillin (CARB) and 30
g/mL chloramphenicol (CAP).
1.
Incubate E.coli bacteria that have been stored in LB culture medium containing 20 % glycerol at 70C, at a petri dish containing LB culture medium, agar, CARB and CAP at 37C for about 24
hours.
2.
Select one colony for overnight incubation in 150 mL LB culture medium added CARB and CAP,
at 37C in a shaker at 230 rpm.
3.
Distribute the overnight culture equally in five 5 L erlenmeyer flasks containing 1.5 L LB culture
medium added CARB and CAP.
4.
Mount the five flasks in a shaker and incubate at 37C and 200 rpm until the optical density reach
a value of 0.5-0.6.
5.
Add IPTG from a freshly prepared stock solution that has been filtered with a Millex-GP 0.22 m
unit to a final concentration of 0.5 mM in each flask.
6.
Harvest the bacteria after 4 hours of further incubation by centrifugation at 5000 rpm (JA10), 4C
for 10 minutes.
7.
Suspend the bacteria paste in buffer A (2.5 mL buffer A/ gram bacteria paste), distribute the
suspension in 50 mL centrifuge tubes and freeze in liquid nitrogen.
8.
Store frozen bacteria at -74C.
19
Chapter 3
Methods
3.2 Purification of Mouse R2 Protein
All manipulations, mainly carried out as described by Mann et al.,69 were performed
in a cold room (4C) or in a refrigerated centrifuge to avoid protein denaturation.
3.2.1 Lysis of Bacteria
Principle:
Lysozyme, constitutivelly produced in the bacteria, was able to leak out when thawing
the bacteria. Thus the peptidoglycan layer protecting the bacteria were broken down.
Procedure:
1.
Thaw bacteria in a water bath at 5-10C.
2.
Centrifuge at 20 000 rpm (JA25.50), 4C for 1.5 hours.
3.
Collect the supernatant (and measure the volume) for further purification of R2 protein.
3.2.2 Precipitation of DNA
Principle:
Streptomycin sulfate precipitate DNA.
Procedure:
1.
Prepare a 10% (w/v) streptomycin sulfate solution (volume = ¼ of supernatant).
2.
Adjust the pH to 7.0 by adding ammonia (NH3 (aq)).
3.
Add the streptomycin solution drop by drop over 10 minutes to the supernatant from 3.2.1 while
stirring carefully.
4.
Stir carefully for 15 minutes.
5.
Centrifuge at 15 000 rpm (JA25.50), 4C for 20 minutes.
6.
Collect the supernatant (and measure the volume) for further purification.
20
Chapter 3
Methods
3.2.2 Precipitation of R2 Protein with Ammonium Sulfate
Principle:
Proteins have polar surfaces and interact with water mainly through hydrogen bonds
and other dipolar interactions. Water molecules are arranged in a specific manner
around the solvated proteins, and a chaotropic salt as (NH4)2SO4 (ammonium sulfate)
disrupts the matrix formed by the water molecules. This is because chaotropic salts
attract water for their own hydrating shells, and above a certain salt concentration
there is not sufficient amounts of water left to hydrate the proteins. The most
energetically favorable interactions are then found between the proteins themselves,
thus large aggregates of native proteins precipitate. This “salting out” effect is
described by A.A. Green.72
Procedure:
1.
Add 0.243 g (NH4)2SO4/ mL supernatant (about 40% saturation) during a period of 15 minutes
while stirring carefully.
2.
Stir carefully for 30 minutes.
3.
Centrifuge at 10 000 rpm (JA14), 4C for 50 minutes.
4.
Dissolve precipitate in 2-3 mL buffer A.
5.
Add 20 L 10 mM phenylmethylsulfonyl fluoride (PMSF) dissolved in ethanol and mix carefully.
3.2.3 Gel Filtration Chromatography
Principle:
Ion exchange chromatography was used as one of the purification steps. When the ion
strength in the protein solution is to high, the protein will not bind to the column
material. Therefore the salt has to be removed by applying the protein solution at a
G25 column.
Gel filtration chromatography separates molecules by their size.73 The column
material (G25) is composed of small spherical particles with pores, and large
molecules such as proteins are not able to penetrate the particles while small
molecules like salts enter the pores. This results in a longer traveling distance for
small molecules than for large molecules. Large molecules such as proteins will be
eluted in the solvent used to equilibrate the column.
21
Chapter 3
Methods
Procedure:
1.
Equilibrate a 30 mL G25 column with 150 mL buffer A.
2.
Apply the dissolved (NH4)2SO4 precipitate (~3.5 mL).
3.
Elute with buffer A and collect fractions with A280
4.
Wash the G25 column with 300 mL buffer A.
nm
> 0.5.
3.2.4 Anion Exchange Chromatography
Principle:
Solvent exposed protein surfaces are highly populated by polar and charged amino
acid sidechains. Negatively charged sidechains will bind to a positively charged
column material at low ionic strength.73 Variations in the populations of negatively
charged sidechains at protein surfaces among proteins result in selective affinity
between the proteins in the solution and the column material.
At high ionic strengths, counterions of the buffered solution will replace the proteins
that are subsequently eluted.
Procedure:
1.
Degas 7.0 g of the weak anion exchanger DE52 (dietylaminoetyl) dissolved in stock solution A for
30 minutes.
2.
Transfer the degassed column material to a 10 mL column and equilibrate with 200 mL degassed
buffer B.
3.
Apply fractions containing desalted protein collected from the G25 column.
4.
Wash with degassed buffer B (10-15 mL) until A280 nm< 0.1.
5.
Elute with buffer C and collect 5 mL fractions until A280 nm< 0.4.
3.2.5 Sodium Dodecyl Sulfate Polyacrylamide Electrophoresis
All fractions collected from the anion-exchange column with A280> 0.4 was analyzed
by sodium dodecyl sulfate polyacrylamide electrophoresis (SDS-PAGE) with respect
to the purity of R2. The Pharmacia PhastSystem and was used for this purpose.
Principle:
SDS-PAGE separates denatured macromolecules by their molecular mass.74,75
22
Chapter 3
Methods
Procedure:
1.
Mix 14 L protein solution with 8 L Phast loadmix in 1.5 mL eppendorf tubes.
2.
Seal the eppendorf tubes and boil for 10 minutes. Spin at 12000 rpm in micro centrifuge.
3.
Prepare the PhastSystem instrument with Phast gel 8-25% and PhastGel buffer strips and set the
temperature to 16C.
4.
Use the application comb to position 1 L of each prepared sample above the gel in the instrument.
5.
Start the PhastSystem instrument and prepare the development unit of the instrument for staining
(Phast staining solution), destaining (Phast destaining solution), and preservation (Phast
preservation solution) of the gel.
6.
Develop the gel and collect those fractions containing pure mouse R2.
3.2.6 Ultra Filtration
Fractions collected in step 3.2.5 had a concentration of about 1 mg/mL. Further
experiments required protein concentrations of about 50 mg/mL, thus a concentration
of the protein solution was necessary.
Principle:
The protein is concentrated using a collodion bag with molecular weight cutoff
(MWCO) 12000 D attached to a vacuum pump extracting the solvent, salt, and buffer
molecules through the semi-permeable membrane leaving the protein inside the bag.
Procedure:
1.
Equilibrate the collodion bag in Milli Q filtered and ion-exchanged H2O (mqH2O) for 1 hour.
2.
Mount the vacuum dialysis system in the cold room and start the system with only buffer A inside
the collodion bag.
3.
Remove excess of buffer A after 5 minutes of dialysis and apply the fractions that passed the SDSPAGE control.
4.
Let the system run overnight, dissolve concentrated protein in 200 L of buffer D, and freeze in
liquid nitrogen. Store at -74C.
23
Chapter 3
Methods
3.3 Protein Quantification
3.3.1 Quantification Using UV/vis Spectrophotometry
The extinction coefficient (280 -310 nm = 124000 M-1cm-1) for the apo mouse R2 dimer
at 280 nm was determined in a spectrophotometric experiment from the A280 nm – A310
nm
value and the amino acid composition of mouse R2 in 1991.69 This obtained value
was used to quantify the R2 concentration.
Principle:
Aromatic amino acid sidechains, especially tyrosine and tryptophan, absorb UVradiation at 280 nm. This is due to excitation of  (bonding) orbital electrons to *
(antibonding) orbitals.
Procedure:
1.
Record a baseline of buffer A at the spectrophotometer.
2.
Dilute 5 L concentrated apo mouse R2 to 500 L with buffer A.
3.
Measure A280
cR2 
nm
and A310
nm
and calculate the protein concentration in molar.
A 280nm310nm
124000M 1cm 1  1cm
3.3.2 Bio-Rad Protein Assay
The Bio-Rad protein assay (Bradford assay76) was occasionally used as a second
method for determining protein concentrations.
Principle:
Coomassie Brilliant Blue G-250 is stabilized in its anionic form when interacting with
basic and aromatic amino acid sidechains in proteins. The anionic form has an Amax at
595 nm in a spectrophotometric experiment while other forms of the dye that do not
bind to proteins absorb light at other wavelengths.
Procedure:
The procedure for the Bio-Rad protein assay that was obtained from Bio-Rad is well
described in the accompanying manual.
24
Chapter 3
Methods
3.4 Buffer Exchange and Ultra Filtration
Buffer exchange for small sample volumes were carried out by using 1 mL spin
columns.
For larger sample volumes, NAP-5 or PD-10 (G25) columns (from Pharmacia) were
used for buffer exchange. If necessary, the protein concentration was increased by
ultra filtration (Centricon, MWCO 50.000 D). The procedures were described in detail
in the datasheets accompanying the different kits.
3.5 Reconstitution of the Diiron-Oxygen Cluster and the Tyrosyl
Radical
Several procedures for the reconstitution of the diiron-oxygen cluster and the tyrosyl
radical in E.coli and mouse R2 have been described in the literature. A novel
approach, based on a method described for E.coli R229,77,78 was applied to the mouse
R2 protein.
Principle:
The apo mouse R2 protein reacts with two ferrous atoms and dioxygen as described in
the introduction (see Chapter 2.2.2). Oxidation of Fe2+ at low pH is kinetically
forbidden. This allows addition of an acidic aerobic Fe2+ solution to the protein
solution without unnecessary loss of Fe2+.
Procedure:
1.
Exchange protein solvent to buffer D as described in (Chapter 3.4) and quantify the concentration
of R2 protein in the solution (Chapter 3.3.1and 3.3.2).
2.
Prepare a Fe2+ solution with [Fe2+] = 150 [R2] by dissolving ammonium iron(II) sulfate
hexahydrate in mqH2O with pH adjusted to ~2.3 by concentrated nitric acid (HNO 3).
3.
Add the Fe2+ solution to the protein solution until a final [Fe2+] seven times higher than [R2] is
obtained and mix carefully. After a few seconds, the protein solution will turn green indicating a
successful reconstitution of the tyrosyl radical and the iron-oxygen cluster.
4.
Blow O2(g) over the surface of the protein solution for 5 minutes to ensure that there is a sufficient
amount of O2 present in the solution for the reconstitution reaction. This step is especially
important while working with high protein concentrations.
25
Chapter 3
Methods
3.6 Quantification of Dithionite
Disodium dithionite (DT) is a strong reductant with a midpoint potential at pH 7 Em’
= - 420 mV.79 DT was used to reduce an electron transfer mediator, which in turn
reduced the tyrosyl radical and the iron-oxygen cluster. It was very important to
control the amount of primary reductant in the redox system, thus stock solutions of
DT were freshly prepared and quantified before each experiment.
Principle:
Fe(CN)63- has an extinction coefficient of 420 nm,ox = 1040 M-1cm-1 while the value
for the reduced form, Fe(CN)64-, is lower (420 nm,red = 2 M-1cm-1).80 Monitoring the
change in absorbance at 420 nm when adding DT to the Fe(CN)63- solution makes us
able to determine the concentration of DT in the stock solution.
Procedure:
1.
This procedure works for DT stock solutions in the range of 2-100 mM.
2.
Dissolve the DT salt in 200 mM HEPES, pH = 7.5 in sealed vials.
3.
Record a baseline of a cuvette containing 200 mM HEPES, pH = 7.5.
4.
Add small amounts of the DT solution to the sealed cuvette containing 1.0 mL 2.00 mM Fe(CN) 63-,
200 mM HEPES, pH =7.5 until A420 nm ~1.3. There is no oxygen left inside the sealed cuvette at
this point.
5.
Then add 3-8 L, depending of [DT], of the DT solution with a 10 L gas tight syringe to the
cuvette, mix well and read of the A420 nm value.
6.
Calculate the concentration of DT in the solution by the formula
c DT 
AVFe ( III)
2 420VDT 1cm
, where A is the change in absorbance and the volumes VFe(III) (volume
of the Fe(CN)63- solution) and VDT (volume of the DT solution) are given in L.
7.
Repeat the procedure three times to determine the DT concentration properly.
3.7 Anaerobic Reduction of Phenazine Methosulfate
The electron transfer mediator phenazine methosulfate (PMS) was used as the primary
reductant and redox buffer when reducing the tyrosyl radical and the diiron-oxygen
26
Chapter 3
Methods
cluster. It was important to understand the properties of the PMS redox buffer when
studying the equilibrium of the mouse R2 diiron-oxygen cluster oxidation states in a
PMS/ DT solution.
Principle:
When the PMS salt is dissolved in water, a peak in the light absorbance spectrum at
388 nm originating from oxidized PMS can be observed (Figure 3.1). By monitoring
the decrease of the 388 nm peak when known amounts of DT is added, the redox
buffering properties of PMS can be elucidated.
Procedure:
388 nm
PMS is light sensitive and decomposes when
exposed to light with wavelengths below 500
nm. Thus, all manipulations were carried out in
a dark room, and all equipment used for
Absorbance
1.5
1.0
0.5
handling the PMS solution was wrapped in
0.0
aluminum foil. All spectra were recorded using
a diode array spectrophotometer.
1.
Prepare a 38 M PMS solution and a 3.8 mM DT
350
400
450
Wavelength (nm)
Figure 3.1 Absorption spectrum of
oxidized phenazine methosulfate.
solution, both buffered with 50 mM Tris-HCl pH =
7.5, anaerobically.
2.
Quantify the DT soulution as described in Chapter 3.6.
3.
Evacuate a 2 mL quarts cuvette sealed with a rubber septum and flush with Ar (g).
4.
Transfer 1 mL of the anaerobic PMS solution to the cuvette using a 1 mL gas tight syringe flushed
with Ar (g).
5.
Record the spectrum of the oxidized form of PMS before adding 1-10 L DT solution with a gas
tight 10 L Hamilton syringe. Shake the cuvette and record spectrums at 10 seconds intervals until
equilibrium between DT and PMS is reached.
3.8 Reduction of the Diiron-Oxygen Cluster and the Tyrosyl Radical
The ferric irons in reconstituted mouse R2 are antiferromagnetically coupled. This
results in a S = 0 state for the diiron-oxygen cluster. For EPR spectroscopy, it is
necessary to have a paramagnetic half integer or integer spin system. However, it is
27
Chapter 3
Methods
possible to reduce the diiron-oxygen cluster to a mixed valence state that has a ground
state S = ½ spin system that is EPR active.
Principle:
Electron transfer mediators are often employed to transfer electrons from either a
working electrode or a chemical reductant the metal cluster(s) in the protein
examined.
Procedure:
All manipulations are carried out at 0-4C.
1.
Bubble 100 mL of 50 mM HEPES, 100 mM KCl, 20% glycerol, pH = 7.5 with O 2 free Ar (g) for
one hour to remove O2(aq).
2.
Reconstitute the protein in a glass vial as described in 3.5 before sealing with a butyl septum.
3.
Evacuate and flush the sealed vial with O2 free Ar (g) at least 10 times, and leave under 2-3 Bar
Ar(g) when finished.
4.
Prepare two 1 mL glass vials with proper amounts of DT and PMS, respectively, seal with butyl
septa and carry out 5 cycles of evacuation and Ar (g) flushing.
5.
Inject 0.5 mL anaerobic HEPES buffer prepared in step 1 applying a 1 mL gas tight syringe. Be
aware that solvated PMS is photosensitive and must be protected from light.
6.
Mix a proper amount of PMS solution into the reconstituted protein solution anaerobically with a
gas tight Hamilton syringe.
Sample Preparation for the Redox Equilibrium Studies
Add DT solution directly into the glass vial containing the anaerobic protein-PMS
solution, or use gas tight Hamilton syringes equipped with long needles and mix the
solutions simultaneously in an EPR tube. Final concentrations of R2, DT, and PMS in
the EPR tubes: [R2]= 50 – 100 M, [PMS]= 2 – 5 mM, [DT]= 0.12 – 1.65 mM.
In the introductory studies of the interactions between the mixed valence cluster and
alcohols, a 50%-50% alcohol – water mixture was added to the samples from the
kinetic studies and incubated at 4 C for 1 minute.
Sample Preparation for the Alcohol Titrations
Add 20 L of 50%-50% alcohol (methanol, ethanol, propanol, and butanol) – water
mixture to each of the EPR tubes and set up the homebuilt EPR sample mixer with
N2(g), a 1 mL gas tight syringe with protein-PMS and a 1 mL syringe with DT
28
Chapter 3
Methods
solution. The N2 (g) flow in the homebuilt EPR sample mixer keep the syringes in an
anaerobic atmosphere and removes the air from the EPR tube that is to be filled.
Final concentrations of R2, DT and PMS in the EPR tubes: [R2]= 100 M, [PMS]= 5
mM, [DT]= 1.7 mM.
3.9 Electron Paramagnetic Resonance Spectroscopy
3.9.1 Introduction to EPR Theory
Magnetic resonance is the phenomenon characterized by the change of the sign of a
spin experiencing a static magnetic field when absorbing electromagnetic radiation.
Electron paramagnetic resonance (EPR) spectroscopy is a magnetic resonance
technique not very different from nuclear magnetic resonance (NMR). With NMR the
nuclear magnetic moments of atoms with nuclear spin I  0 are detected, while the
magnetic moments of unpaired electrons in radicals and metal ions are detected by
EPR.
An electron is an elementary particle that is characterized by its mass, charge, and
angular momentum quantum numbers (Table 3.1).
Table 3.1 Angular momentum quantum numbers81
Name
Representation
Value
Orbital angular momentum a
L
0, 1, 2…
Orbital magnetic a
Ml
0,  1,  2,…,  L
S
0,
Ms
0,  12 ,  1,  32 ,  S
Spin angular momentum
Spin magnetic a
a
1
2
, 1, 32 , 2. . .
a
Upper case letters are used for orbital and spin angular momentums larger than 1 and ½, respectively,
else lower case letters are used.
In general, spin and orbital angular momentum quantum numbers are eigenvalues of
the eigenvalue equation Ĵ z   jz  , where Ĵ z is the general spin or orbital angular
momentum operator (operators always have ^ “hats” in this thesis), jz is the
eigenvalue (quantum number), and  the eigenfunction of Ĵ z .82 Later in this section,
the spin Hamiltonian operator ̂ will be described, hence the operator representation
29
Chapter 3
Methods
of spin and orbital angular momentum will be used instead of quantum numbers
throughout the text. Information obtained from an EPR spectrum is usually analyzed
both qualitatively and quantitatively. By considering the positions, shapes, and
linewidths of the peaks in the EPR spectrum, preliminary information about the spin
system is obtained. Further analysis usually involves quantum mechanical
calculations using a phenomenological spin Hamiltonian operator at a set of
wavefunctions in order to obtain possible energies for the transitions observed in the
experimental EPR spectrum. Thus, phenomenological spin Hamiltonians are used to
parameterize experimental EPR spectra. By simulating an experimental EPR
spectrum, physical properties can be assigned to the paramagnetic EPR active spin
center. Parameters for the spin Hamiltonian used in this thesis are described below,
and their values can be obtained by simulating EPR spectra.
3.9.2 The Electronic Zeeman Effect
Classically, the circulation of a charge results in a magnetic field, and the zcomponent of the magnetic moment of the electron represented by the operator ̂ z
can be explained by considering the rotation of a point charge. Though the classical
explanation is not entirely correct, the magnetic moment from the classical model is a
factor 2 lower than expected from quantum mechanics, it is still a reasonable
illustration.
In a static magnetic field, the z-component of the magnetic moment vector of the
electron can be oriented either along or against the field, and this phenomenon is
termed the electronic Zeeman effect. The energy level difference between these two
states is very small, and both states will be populated almost equally. Due to the small
population difference between the two energy levels, an EPR detectable net magnetic
moment appears. The population difference is described by the Boltzmann
distribution and depends on the temperature T and the separation of the energy levels
E as shown in Equation 3.1.82
 E
Ni
 e k bT
Nj
Eq. 3.1
30
Chapter 3
Methods
Ni and Nj are the populations of spin aligned along or against of the field direction,
respectively, and kb is the Boltzmann constant.
The spin Hamiltonian for a free electron in a static magnetic field is given by:
ˆ  Bˆ   BŜ  Bg Ŝ

e
z
e
z
e z
Eq. 3.2
where B is the magnitude of the static magnetic field, e the electron gyromagnetic
ratio,  the Bohr magneton, and Ŝ z the z-component of the electron spin operator.82
The energy levels for a free electron in a magnetic field are obtained by letting ̂ e
operate at the electron spin functions |+1/2 and |-1/2 as illustrated in Equation 3.3
and 3.4 .82 This function annotation is termed Dirac notation, where < f | is the
complex and | f > the real function.
Useful relations
e  
ge | e |
, where e is the elementary charge, me the electron mass, and ge the free electron g-factor.
2m e
h
, where h is the Planck constant.
2
|e|

2m e

ˆ | 1 / 2   Bg Ŝ | 1 / 2   1 / 2Bg | 1 / 2 

e
e z
e
Eq. 3.3
ˆ | 1 / 2   Bg Ŝ | 1 / 2   1 / 2Bg | 1 / 2 

e
e z
e
Eq. 3.4
Equations 3.3 and 3.4 can be written as:
E = + ½ Bge
Eq. 3.5
E = - ½ Bge
Eq. 3.6
E and E given in Equation 3.5 and 3.6 are the energy levels of the electrons where
the z-components of the spins are oriented against and along the field, respectively. In
order to achieve electron paramagnetic resonance, the electrons have to absorb
31
Chapter 3
Methods
microwave photons with the discrete energy E = E - E. This results in the
formulation of the resonance condition;
E = E - E = h
Eq. 3.7
where  is the microwave frequency.82
In EPR, the magnetic field (B) is
+ ½ Bge
E = h
E
- ½ Bge
varied while the frequency is kept
constant. The electronic Zeeman
effect is illustrated in Figure 3.2,
B=0
Increasing B
where the resonance condition is
fulfilled when the magnitude of B
reaches a certain level.
Figure 3.2 The energy levels E and E increase and
decrease with slopes of + ½ Bge and - ½ Bge,
respectively.
The g-value of the free electron ge is 2.002319. Observed, effective g-values usually
deviate from ge, and especially in the case of transition metals. This can be explained
by:
1. The g-values are related to the splitting of the d-orbitals of a coordinated metal
atom.83
2. Modulations of the ligand field due to molecular vibrations that disturb the
electronic environment (this is the Jahn-Teller effect, which will not be further
discussed in this thesis).83
The symmetry of a spin center is reflected in its EPR signal g anisotropy, and some
examples of symmetries giving characteristic EPR signals is illustrated in Figure 3.3,
adapted from Hanson and Solomon.67
32
Chapter 3
Methods
Figure 3.3 Different geometries of coordinated transition metal ions found in metallo proteins; A;
pure octahedral (gx = gy = gz), B; rhombic (gx  gy  gz), C; square planar (gx = gy  gz), D and E;
distorted tetrahedral (gx  gy  gz).
The Hamiltonian for a free electron in Equation 3.2 is not adequate for describing
complicated spin systems where the orientation of the spin system to the magnetic
field and the orbital distribution of electrons have to be considered. In order to include
anisotropy, the electronic term of the spin Hamiltonian is written as

Ĥ e   B ~
gŜ
Eq. 3.8

where the static magnetic field is given by the vector B , the g values is given in the
3x3 tensor ~g with the diagonal as the principal g-values (gx, gy, gz), and the spin
operator Ŝ .
3.9.3 Zero Field Splitting
For transition metals with S > ½, interactions between the electrons themselves within
the metal atom contributes to additional splittings of the Zeeman energy levels. The
origin of this effect is the electron-electron interaction that is induced by the metal
ligands and mediated by the spin orbit coupling.83 By decreasing the temperature, the
ground state get more heavily populated, and the influence of the exited states
mediated through the spin orbit coupling will change. Thus a temperature dependence
of this type of splitting can be observed. The spin Hamiltonian term for this
phenomenon is given as
~
Ĥ ZFS  ŜDŜ
Eq. 3.9
33
Chapter 3
Methods
~
where D is a traceless, symmetric 3x3 tensor. Since the magnetic field is not included
in this term, the splitting of the energy levels is independent of the static magnetic
field, hence it is called the zero field splitting (ZFS) term.
Equation 3.9 can be rewritten to
1


Ĥ ZFS  D Ŝ z  S(S  1)  E(Ŝ 2x  Ŝ 2y )
3


where S is the total spin and
Eq. 3.10
E 1
 . The D and E-values are named the axial and
D 3
rhombic zero field splitting parameters, respectively.84 By considering Equation 3.10,
it can be noticed that the D-value describes ligand-induced symmetry distortions
along the z-axis of the spin system, while the E-value describes the deformations
along the x and y-axis. High spin non heme ferric irons usually have |D|-values below
1 cm-1 while high spin non heme ferrous irons have much higher values (|D| 5 – 15
cm-1).
3.9.4 Exchange Coupling
Dinuclear metalloproteins usually have bridging ligands linking the metal atoms
together. Such a bridging ligand couples the two spins (Sa and Sb) either
ferromagnetically or antiferromagnetically depending on the nature of the bridge.
When coupled antiferromagnetically, the spins are added together producing
Stotal = Sa + Sb, Sa + Sb –1, Sa + Sb – 2, . . . , |Sa - Sb|
Eq. 3.11
spin states where |Sa - Sb| has the lowest energy. The Heisenberg, Dirac, Van Vleck
(HDVV) exchange Hamiltonian for a coupled spin system is given by
ˆ  2JŜ Ŝ

ex
a b
Eq. 3.12
34
Chapter 3
Methods
where J is the Heisenberg coupling constant.84 By solving Equation 3.12 the energy
levels for the spin states calculated in Equation 3.11 can be obtained (Equation 3.13).
ES-total = -J[Stotal(Stotal + 1) – Sa(Sa + 1) – Sb(Sb + 1)]
Eq. 3.13
3.9.5 The Spin Hamiltonian for a Dinuclear Coupled Metal Cluster
To this, point several spin Hamiltonian terms have been presented. In order to
describe the paramagnetic properties of a dinuclear coupled metal cluster these terms
must be summed up. The electronic spin Hamiltonian (from the Zeeman effect) and
the zero field splitting spin Hamiltonian are defined for each of the atoms while the
exchange Hamiltonian link the two spin systems together as in Equation 3.14.83


~
~
ˆ  2JŜ Ŝ  B~

ga Ŝa  B~
g bŜb  Ŝa Da Ŝa  Ŝb D bŜb
a b
Eq. 3.14
3.9.6 Relaxation of a Paramagnetic System
An important aspect of EPR is the way a spin system is excited by absorption of
microwaves and how it relaxes again to reach thermal equilibrium. Equation 3.1
describes the distribution of ms = + ½ and ms = - ½ in a static magnetic field, and N
= Ni – Nj is the number of electrons contributing to the net magnetic moment. When
the resonance condition (Equation 3.7) is satisfied, electrons in the ms = + ½ or ms = ½ state will change the sign of their spins. Because the probability for changing spin
sign is equal for both states, N will approach 0 when the absorption of microwave
photons occur at a higher rate than the relaxation back to the equilibrium given in
Equation 3.1. When N decrease, the magnetic moment also decrease in magnitude,
and the EPR signal disappears.
The spin system is characterized as unsaturated when the spin relaxation is faster than
the microwave absorption and saturated when the microwave power is high enough to
overcome the spin relaxation.83 The double integral of the EPR first derivative
spectrum is proportional to the square root of the microwave power when the spin
35
Chapter 3
Methods
system examined is unsaturated, and under such conditions the amount of spins of
different unsaturated paramagnetic species can be compared.
The relaxation rate is usually described by the spin-lattice (T1) and spin-spin (T2)
relaxation times. Spin-lattice relaxation is mainly due to vibrational interactions
between the paramagnetic cluster and the lattice while spin-spin relaxation involves
interactions between two or more paramagnetic centers. Pure spin-lattice relaxation
results in homogeneously broadened Lorentzian shaped EPR absorption lines. When
relaxation is mediated through both spin-spin and spin lattice interactions, the EPR
absorption lines are inhomogeneously broadened and a Gaussian lineshape is
expected.83
For a complex paramagnetic system such as a bridged dinuclear cluster, the relaxation
theory is complicated. Because both atoms in the dinuclear cluster are paramagnetic,
distance and orientation dependent spin-spin interactions are expected to contribute to
the relaxation of the paramagnetic system in addition to spin-lattice interactions.83
36
Chapter 3
Methods
3.9.7 EPR Instrument Parameters Used in the Experiments
Different parameter sets were used, depending on what kind of sample investigated.
The parameters and their applied values are described briefly in Table 3.2.
Table 3.2 EPR instrumental parameters
Parameter
Description
Frequency
Microwave frequency (9.6 GHz)
Power
Microwave power (200 nW – 200 mW)
Sweep width
The magnetic field sweep range (0-5000 G).
Center field
The magnetic field is swept about this value (0-5000 G).
Resolution
Number of data points collected (1024 ).
Receiver gain
Amplification of detector signal (2103 - 1106).
Modulation frequency
The frequency which the magnetic field is modulated (100 kHz)
Modulation amplitude
Amplitude of the field modulation (3 – 9 G).
Modulation phase
Detection phase of the detector (90 degrees)
Conversion time
Time to convert analog voltage to digital value (81.92 ms).
Time constant
Time to filter the analog signal (40.96 ms).
Sweep time
Time to record spectrum (83.89 s).
Harmonic
Selection between first or second derivative detection mode (1 or 2)
3.9.8 EPR Sample Preparation
An anaerobic EPR sample is either prepared inside a sealed anaerobic EPR tube or
transferred into a tube with a long-needle gas tight Hamilton syringe. The EPR tubes
used have the dimensions 25 cm x 3.8 mm (inner diameter), and the sample volume
were in the range between 180 and 200 L.
Prepared samples are sealed with butyl septa and stored in N2 (l) at 77 K.
3.9.9 Quantification of Spin in an EPR Sample
A standard procedure was used to determine the quantities of unpaired electrons.
Typically, the first derivative of the EPR absorption curve is recorded. Thus enhanced
resolution of the spectra is achieved. The double integration of the EPR first
37
Chapter 3
Methods
derivative absorption spectra acquired for an unknown sample can be compared to
that of a standard, and the amount of unpaired electrons in the unknown sample can
be determined. A 1 mM Cu(II) standard, from which a S = ½ EPR signal can be
recorded, was used for all spin quantifications.
3.10 Circular Dichroism Spectroscopy
In Circular Dichroism (CD) spectroscopy the difference in absorbance between left
circularly polarized and right circularly polarized light is detected. Thus, this
technique can be used to study chiral molecules.85 The origin of the CD effect is that
in symmetrical molecules the electronic and magnetic transition dipoles are
perpendicular to each other, while that is not the case in asymmetric molecules. The
rotational strength (the change in the extinction coefficients Left and Right as functions
of the wavelength), which is measured in CD spectroscopy, depends on the angle
between the electronic and magnetic transition dipoles.
CD is expressed as either the difference in absorbance between the left and the right
circularly polarized ligth A = ALeft – ARight or the ellipticity  that represents the
rotational strength. The relationship between A and  is given by A = / 32.98.
3.10.2 CD instrumental parameters
Table 3.10.1 Parameters used for the CD experiments
Parameter
Value
Band with
1 nm
Response
1s
Sensitivity
Standard
Measurement range
460 – 290 nm
Data pitch
0.5 nm
Scanning speed
50 nm/ min
Accumulation
1
Temperature
4 C
38
4 Results and Analysis
4.1 Protein Purification
The mouse R2 protein purification procedure, which is described in Chapter 3.1 and 3.2,
was published by Mann et al.69 in 1991. This procedure has routinely been carried out in
our laboratory since 1994, and the protein yield and purity are mainly the same for all the
purifications. For all EPR and CD sample preparations, the protein purity was considered
to be higher than 95 % by visual inspection of protein samples separated by SDS-PAGE.
The results from the experiments involving protein purification (Chapter 3.1 and 3.2),
protein quantification (Chapter 3.3) and solvent manipulations (Chapter 3.4) will not be
further discussed.
4.2 Reconstitution of Mouse RNR-R2
A novel approach was chosen for the reconstitution of the diiron-oxygen cluster and the
tyrosyl radical in mouse R2. The motivation for exploring new reconstitution techniques
was to save time and reduce the risk of adding oxidized iron (Fe3+) to the apo-protein
solution. The method previously reported for mouse R2 was based on initial binding of
ferrous (Fe2+) in the protein in the presence of ascorbate before molecular oxygen was
added.69 In the case of E.coli R2 protein reconstitution, O2 was already present in both
the protein solution and the acidic ferrous iron solution,29,77,78 or Fe2+ and ascorbate were
added anaerobically.10 We based our novel routine on an aerobic approach utilizing an
acidic Fe2+ solution. The tyrosyl radical yields that were reproducibly obtained are
presented in Table 4.1 and compared to published results for both mouse and E.coli R2.
39
Chapter 4
Results and Analysis
Table 4.1 Results from the reconstitution of mouse R2
Method
Tyrosyl radical per R2 dimer
Fe2+ and ascorbate added anaerobically (mouse R2) 69
2+
Fe and ascorbate added anaerobically (E. coli R2)
2+
Acidic Fe solution added aerobically (E. coli)
2+
1.2 – 1.5
1.2
1.2  0.1
78
Acidic Fe solution added aerobically (mouse R2)
a
10
1.7  0.1
a
This work.
4.3 Redox Studies of Phenazine Methosulfate
4.3.1 Purpose of the Experiments
The electron transfer mediator phenazine methosulfate (PMS) was used when reducing
the diferric cluster and tyrosyl radical in reconstituted mouse R2.
It was important to understand the redox buffering properties of PMS before starting
upcoming experiments.
The redox chemistry of PMS is
complex. Different forms of PMS are
illustrated in Figure 4.1, where PMS+
(the oxidized cationic form) and
PMSH (the two electron reduced,
protonated form) dominate at pH
7.5.86 The one electron reduced
semiquinoid forms PMS and
PMSH+ are only stable at pH < 3.5,
and only small amounts are detected
Figure 4.1 Phenazine methosulfate (PMS) can have
at pH 7.5.86,87 When both PMS+ and
different protonation and redox levels.
PMSH are present in the same solution, formation of a complex (PMSHPMS)+ that is
not reduced by NADH (nicotinamide-adenine dinucleotide) has been suggested.86,87 The
40
Chapter 4
Results and Analysis
equilibrium of the proposed complex formation where Keq = (1.3  0.2) 104 M-1,86 is
illustrated in Scheme 4.1.
eq
PMS   PMSH 

(PMSH  PMS) 
K
Scheme 4.1
4.3.2 The Equilibrium between Different Redox Forms of PMS
The spectral changes observed when two reducing equivalents per PMS+ molecule were
added to an anaerobic PMS+ solution are shown in Figure 4.2. Small amounts of DT were
added after virtually full
reduction of PMS+ was achieved,
that spectrum B in Figure 4.2
represents PMSH. When 0.5, 0.8,
1.0, 1.6 reducing equivalents per
Absorbance
spectrum appeared. This indicates
0.8
0.75
A
A388 nm
and no further changes in the
0.50
0.6
0.4
0.2
0.5
1.0
1.5
2.0
Reducing equivalents
0.25
B
PMS+ were added, a linear
relationship between the decrease
350
of A388 nm (A388 nm) and the
400
450
500
Wavelength (nm)
amount of reductant was observed
Figure 4.2 Anaerobic reduction of 38 M PMS by
38 M dithionite in 50 mM Tris at pH 7.5. Spectra
A and B are fully oxidized and fully reduced PMS,
respectively. Inset: the decrease of A388 nm as a
function of reducing equivalents added.
(Figure 4.2, inset). The initial
concentration of PMS+ was
quantified using the extinction
coefficient PMS+ = 26.3 mM-1 cm-1.86 DT solutions were quantified using the method
described in Chapter 3.6 before and after the experiment.
Semiquinoid forms of PMS have absorption maximums different than PMS+ and PMSH,
and were not observed at pH = 7.5 by light absorption spectroscopy. An intense EPR
signal originating from the PMS semiquinoid forms was observed in the samples
containing mouse R2 mixed valence cluster. However, the amount of semiquinoid PMS
41
Chapter 4
Results and Analysis
forms represented only (0.28  0.06) % of the initial PMS concentration in these samples.
Thus, no significant effect from the semiquinoid forms at the PMS+/ PMSH redox couple
equilibrium should be expected at pH 7.5.
Halaka et al.86 did not observe the equilibrium described in Scheme 4.1 directly. They
assumed that the absorption spectrum of (PMSHPMS)+ was the sum of the PMS+ and
the PMSH absorption spectra. Another assumption made by Halaka et al.86 was that the
(PMSHPMS)+ complex did not react with NADH in the observed time window. Hence,
they could explain that the complete reduction of PMS+ was not achieved when more
than equimolar amounts of NADH were added.86
Our results, however, show a linear dependence between the reduction of PMS+ to PMSH
and the amount of reductant added. Thus, it is reasonable to assume that no
(PMSHPMS)+ complex unable to react with DT is formed and that the redox couple
PMS+/ PMSH dominates the redox equilibrium in a PMS solution when DT is added
under anaerobic conditions.
4.4 Reduction of the Tyrosyl Radical and the Diiron-Oxygen Cluster
4.4.1 Purpose of the Experiments
When studying metal clusters in proteins by spectroscopy, it is important that there is a
certain concentration of the redox state of interest in the sample. The spectroscopic
methods Mössbauer and MCD that are common in bioinorganic chemistry require 1-2
mM protein. EPR is usually more sensitive than Mössbauer and MCD, depending on the
broadness of the EPR signal. Usually, protein concentrations of 50 – 300 M are
sufficient for EPR spectroscopy.
The mixed valence oxidation state of the diiron-oxygen cluster in mouse R2 was the main
research object of this project. This redox form had to be obtained by reducing the
42
Chapter 4
Results and Analysis
reconstituted protein. The previously reported yields of the mixed valence form obtained
by chemical reduction were not high.88,89 This was the initial motivation for the
introductory studies of the redox properties of the diiron-oxygen cluster in mouse R2.
As illustrated in Figure 4.3, the various reduction steps have dissimilar redox potentials.
Tyr-O'
Tyr-OH
Tyr-OH
H
Glu
O
3+
3+
Fe
Fe
2 e-, 2 H
O
+
3+
2+
Fe
Fe
e-, H+
O
O
2+
O
A
O
E1’
Glu
O
B
O
E2’
Fe
O
C
Glu
2+
Fe
O
Glu
Figure 4.3 Different redox states of the diiron-oxygen cluster and the redox active tyrosine in RNR
R2; reconstituted R2 (A), mixed valence form (B) and the fully reduced form (C).
The diferric (Figure 4.3 A) and the diferrous (Figure 4.3 C) form of E.coli R2 have been
characterized by crystallography.8,26 As indicated in Figure 4.3 the ligands of the irons are
partly exchanged when the diiron-oxygen cluster is reduced. Is should be emphasized that
the formal redox potentials E1’ and E2’ describes the redox properties of the irons and not
the thermodynamics of the rearrangement of the iron ligands.
In Figure 4.3, the reduction of the tyrosyl radical and the diiron-oxygen is expressed as
one reaction that is described by the redox potential E1’. This is not an accurate
description of the reaction because the reduction of the tyrosyl radical and the diironoxygen cluster are two independent reactions. The tyrosyl radical have been suggested to
have a redox potential between 700 and 1000 mV.38 Thus, it was assumed that the tyrosyl
radical was reduced prior to the diiron-oxygen cluster and that an equilibrium between
the various oxidation states of the diiron-oxygen cluster and the redox buffer was formed.
Knowing estimates of E1’ and E2’ would able us to increase our yields of the mixed
valence cluster in mouse R2.
43
Chapter 4
Results and Analysis
4.4.3 Estimation of the Midpoint Potential Em’ of the Diiron-Oxygen Cluster
The observation of the mixed valence form of the mouse R2 by EPR made it clear that
the two irons in the diiron-oxygen cluster had individual redox potentials.36
Consequently, the presence of the three redox-states of the diiron-oxygen cluster; the
diferric, the mixed valence, and the diferrous, will depend on the redox potential of the
solution (Esol’).
A simple system for estimating the redox potentials E1’ and E2’ was established. A large
excess of an electron transfer mediator (M) compared to reconstituted R2 was added
anaerobically to the protein solution. The redox potential of the solution (Esol’) was then
adjusted by changing the ratio of the redox couple Mox/ Mred (Mox and Mred are the
oxidized and the reduced forms of the electron transfer mediator, respectively) with the
potent reductant DT. Since [M] was 20-100 times higher than [R2] in all our experiments,
it was assumed that the Mox/ Mred redox couple determined the redox potential of the
solution and that contributions from the diiron-oxygen cluster could be neglected.
The reduced forms of the two electron transfer mediators used in these experiments; PMS
and Toluidine Blue O (TB), are both 2 electron reductants at pH 7.5.90 At pH 7 PMS and
TB have the midpoint potentials (Em’) 85 and 34 mV, respectively.90-92 All the redox
potentials mentioned in this thesis are reported versus the standard hydrogen electrode
(SHE). The midpoint potential of an electron transfer mediator is defined as the redox
potential of the solution when [Mred] = [Mox].
When using the electron transfer mediator PMS, redox equilibrium between the various
redox states of the diiron-oxygen cluster and the electron transfer mediator was reached
within one minute (representative example in Figure 4.4 A). TB was only used in one
experiment, and 15 minutes incubation times for the samples were necessary before the
mouse R2 mixed valence form was stabilized (Figure 4.4 B). The yields of mixed valence
cluster were quantified by double integration of the EPR absorption first derivative
spectra.
44
Chapter 4
Results and Analysis
0.4
Fe(II)Fe(III)/ R2
Fe(II)Fe(III)/ R2
0.4
A
0.2
10
20
B
0.2
30
10
Time (min.)
20
30
Time (min.)
Figure 4.4 Formation of the mouse R2 mixed valence form. The black line indicates the
progression of the reaction. The diiron-oxygen cluster and tyrosyl radical were reduced
by: A; 5 mM PMS, 1.25 mM DT, B; 5 mM TB, 1.25 mM DT. Conditions; 100 M R2
(dimer), 20 % glycerol, 50 mM HEPES pH = 7.5, 100 mM KCl.
Values of Esol’ were calculated using the Nernst equation:
'
E sol
 E 'm 
M ox   RT ln 10 -pH  ln 10 -pHEm
RT
 ln
M red  nF
nF


Eq. 4.1
where R is the gas constant, T the temperature, n the number of electrons involved in the
reaction, F the Faraday constant, [Mox] the concentration of oxidized mediator, [Mred] the
concentration of reduced mediator, and pHEm the pH at which the midpoint potential of
the electron transfer mediator (Em’) had been determined. The last term of the Equation
4.1 equals -15 mV when pH – pHEm = 0.5, n = 2, and one proton is involved in the
reaction.
The Esol’ values and the yields of the mixed valence form obtained using various
concentrations of reactants are listed in Table 4.2.
45
Chapter 4
Results and Analysis
Table 4.2 Calculated redox potentials and mixed valence yields
[R2]a
[Mediator]
[DT]b
[Mox] /[Mred]
E’sol (mV)c
[FeII-FeIII] / [R2]d
100 M
2 mM (PMS)
120 M
15.6
102
0.031  0.001
110 M
2 mM (PMS)
240 M
7.3
93
0.081  0.02
100 M
5 mM (PMS)
1 mM
4.0
86
0.18
100 M
5 mM (PMS)
1.25 mM
3.0
83
0.21  0.2
100 M
5 mM (PMS)
1.5 mM
2.3
80
0.2
50 M
5 mM (PMS)
1.65 mM
2.0
78
0.28  0.1
100 M
5 mM (TB)
1.25 mM
3.0
32
0.22  0.2
a
The concentrations of R2 were determined as described in Chapter 3.3.
b
The DT solutions were quantified (Chapter 3.6) before they were added.
c
The values were calculated form Equation 4.1 and are reported versus the SHE.
d
The amount of mixed valence cluster per R2 monomer (1 is the theoretical maximum value). Each value
is calculated from 3 – 5 data points after redox equilibrium was reached (see representative examples in
Figure 4.4).
In order to estimate the redox potentials E1’ and E2’, it was assumed that only the three
redox states depicted in Figure 4.3; the diferric, the mixed valence, and the diferrous form
were present in the samples. This assumption is supported by the fact that all three
depicted redox states of the diiron-oxygen cluster have been observed by either X-ray
crystallography8,26 or EPR spectroscopy.36,37 The Nernst equations for the different
species were then rearranged such that the fraction of the mixed valence form [FeII-FeIII] /
[R2monomer] was expressed by the redox potentials E1’, E2’, Esol’, and the number of
electrons involved in the first (n1) and the second (n2) electron transfer (Equation 4.2).93
Fe
II
Fe III

R 2 monomer 
1
1  exp (n 1 F / RT )( E' sol E'1 )  exp (n 2 F / RT )( E' 2 E' sol )
Eq. 4.2
The constants in Equation 4.2 have been introduced previously. This equation was used
to estimate E’1 and E’2 from the data listed in Table 4.2. The results from the fitting
processes are shown in Figure 4.5. Because the oxidation of PMSH to PMS+ involves a
two-electron transfer, the best fits of the data in Table 4.2 was obtained with n1 = 2 and n2
46
Chapter 4
Results and Analysis
0.4
= 1 or 2.
mechanism of the electron
diferric cluster. However, it
III
has been demonstrated that
II
transfer from PMSH to the
[Fe Fe ]/ [R2monomer]
We will not speculate on the
0.3
0.2
0.1
the reaction between the
electron transfer mediator
0.0
0
phenazine ethosulfate (Em’ =
55 mV, 2 electron transfer at
pH 790), which is similar to
PMS, and the diiron-oxygen
cluster is reversible.88
40
80
120
E'sol (mV)
Figure 4.5 The data listed in Table 4.3 was fitted with
Equation 4.3, and different values of n1 and n2 were tested.
Dotted (…) line; n1 = n2 =2, solid line; n1 = 2 and n2 = 1,
dashed line (---); n1 = n2 = 1. The arrows indicate the different
midpoint potentials (Em’).
Table 4.3 Estimated redox potentials for the reduction of the mouse R2 diiron-oxygen
cluster (versus the SHE) a
n –values
E1’ (mV)
E2’ (mV)
Em’ b
n1 = 2, n2 = 2
70
43
57
n1 = 2, n2 = 1
71
53
62
n1 = 1, n2 = 1
55
48
52
a
The values are calculated from Equation 4.2 and the plots are shown in Figure 4.5.
Em’ is the midpoint potential given by E1’ + E2’/ 2, and is the redox potential where the highest yield of
the mixed valence form is obtained by the different models.
b
It should be emphasized that these experiments were conducted only to estimate the
redox potential Em’ of the reduction of the Fe(III)-Fe(III) cluster to the Fe(II)-Fe(II)
oxidation state, and that the values presented in Table 4.3 may not be accurate.
The estimates of the Em’ value of the reduction of the diiron-oxygen cluster made us able
to adjust the redox potential of the solution in our samples so that Esol’ ~ Em’. A
maximum yield of the mixed valence oxidation state is obtained when Esol’ = Em’.
47
Chapter 4
Results and Analysis
4.5 Interactions between Alcohols and the Diiron – Oxygen Cluster
4.5.1 Purpose of the Experiments
A novel EPR spectrum was observed when a mouse R2 mixed valence sample containing
5 % (v/v) ethanol was investigated. The novel signal looked like a perturbed native mixed
valence signal and the shifts from the effective (or observed) geff values were large,
indicating an interaction between the mixed valence diiron-oxygen cluster and ethanol.
In order to characterize the possible interaction, various primary alcohols were added to
samples containing mouse R2 mixed valence diiron-oxygen cluster prior to EPR analysis.
4.5.2 Affinity of Various Primary Alcohols to the Mixed Valence Cluster
It was of interest to investigate the effect that selected alcohols had with respect to the
electronic environment surrounding the diiron-oxygen cluster. Primary alcohols with
varying lengths of alkyl chains were chosen for this experiment. Samples containing
mixed valence cluster were incubated for one minute in presence of 1 M (about 5 %
(v/v)) methanol, ethanol, 1-propanol, and 1-butanol, respectively. EPR spectra of the
samples were recorded at 4 K and compared to the native mixed valence signal (Figure
4.6 A-E).
48
Chapter 4
Results and Analysis
EPR is a very powerful technique for probing the electronic structure of a spin center, and
a perturbed EPR signal indicates a change in the electronic structure of a compound.
Consequently, binding of small molecules like alcohols and anions to diiron-oxygen
clusters have been detected by
EPR.2,42,53,54,94,95
gx
eff
eff
gy
eff
As shown in Figure 4.6, all four
gz
alcohols perturbed the electronic
A
structure around the diiron-oxygen
The effective g-values are listed in
Table 4.4. Trends of the perturbations
of the EPR spectra caused by the
alcohols were observed. First, large
shifts in the effective gxeff and gyeff
values, at ~3600 and ~4000 gauss,
respectively, were observed for
B
EPR FIRST DERIVATIVE
cluster in the mixed valence state.
C
D
methanol and ethanol. These gxeff and
E
gyeff shifts were not that pronounced
when 1-propanol and 1-butanol were
added. Second, when the length of
the alkyl chain increased, a shoulder
3600
at ~4000-4150 gauss appeared. This
shoulder is the gzeff peak that overlaps
4000
4400
Field (G)
with the gyeff peak when methanol is
Figure 4.6 Addition of 1 M (about 5 % (v/v) alcohol to
the mixed valence samples. A; native mixed valence signal,
B; 1 M methanol, C; 1 M ethanol, D; 1M 1-propanol, E; 1
M 1-butanol. The samples were incubated with the alcohol
for 1 minute at 4 C. EPR parameters; 5 mW, 4 K, 9.6
GHz.
added and is resolved in the presence
of 1-butanol. From these observed
trends, a preliminary conclusion can
be formulated: the affinity of the
alcohol to the mixed valence cluster decrease with increasing numbers of carbons in the
linear alkyl chain. It was of interest to investigate the nature of the interaction between
49
Chapter 4
Results and Analysis
the alcohols and the mixed valence cluster. Hence it was decided to carry out simulations
of the experimental EPR spectra and relate the results to possible alterations in the
structure of the diiron-oxygen cluster by ligand field calculations (see Chapter 4.6).
Table 4.4 Shifts in effective g-values upon alcohol addition
a
Mixed valence species
gxeff
gyeff
gzeff
Nativ
1.917
1.726
1.600
1 M methanol
1.945
1.743
1.652
1 M ethanol
1.945
1.753
1.660
1 M 1-propanol a
1.938
1.716
1.610
1 M 1-butanol a
1.933
1.735
1.610
Data obtained from a single experiment.
4.5.3 Estimation of Binding Constants for Methanol and Ethanol with Mouse R2
The interesting novel interactions between the selected alcohols and the mouse R2 mixed
valence cluster were further investigated. From the introductory experiments described in
Chapter 4.5.2, it appeared that methanol and ethanol had a higher affinity to the mixed
valence cluster than 1-propanol and 1-butanol did. Therefore, titrations of methanol and
ethanol into solutions of mouse R2 were carried out to observe the alcohol induced EPR
spectrum perturbation. Interestingly, shifts of the g-values were already observed when
50 mM methanol and 100 mM ethanol, respectively, were added to the protein solution.
In the range between 0 and 2 M methanol, the effective gxeff value shifted from 1.92 to
1.95. When the relative change of the gxeff value, grel (Equation 4.3), was plotted against
the concentration of alcohol, the data points formed a hyperbola.
g rel 
gx
eff
 g eff
x , min
Eq. 4.3
eff
g eff
x , max  g x , min
gxeff is the observed gxeff value during the titration, gxeff,min the gxeff value observed for the
native mixed valence signal, and gxeff,max the apparent gxeff value when the diiron-oxygen
cluster is saturated with alcohol.
50
Chapter 4
Results and Analysis
The data points were fitted with an equation describing the binding of a single ligand to a
complex (Equation 4.4, Figure 4.7).96 From this, the binding constant Kb was estimated.
 Alcohol  

g rel  B max 
 K b  Alcohol  
Eq. 4.4
The titration experiment was repeated using ethanol instead of methanol. Equation 4.3
1.0
1.0
0.8
0.8
0.6
0.6
grel
grel
and 4.4 were also applied to the data from this experiment (Figure 4.7).
0.4
0.4
0.2
0.2
0.0
0.0
0.0
0.5
1.0
1.5
2.0
0.0
[MetOH] (M)
0.2
0.4
0.6
0.8
1.0
[EtOH] (M)
Figure 4.7 Titration with methanol and ethanol. The curves are the one site binding equation plots that
was used to estimate the binding constants of methanol and ethanol to the mouse R2 mixed valence
cluster. Fitting parameters: methanol; Chi2 = 0.00096, R2 = 0.992, Bmax = 1.13 and Kb = 0.24  0.02 M.
ethanol; Chi2 = 0.0028, R2 = 0.981, Bmax = 1.6 and Kb = 0.60  0.03 M.
The results from these experiments are summarized in Table 4.5. Our hypothesis, that
alcohols are able to bind in a specific manner to the mouse R2 mixed valence cluster is
supported by our observations.
The observation that Kb, methanol < Kb, ethanol supports the preliminary conclusion presented
in Chapter 4.5.2 that the affinities of the alcohols to the mixed valence cluster decrease
with the length of the alkyl chain. However, these results should be considered
preliminary because the titration experiments have not been repeated.
51
Chapter 4
Results and Analysis
Table 4.5 Estimated binding constants for alcohols to the mixed valence cluster
Alcohol
Concentration range (M)
R2
Kb (M)a
Methanol
0–2
0.992
0.24  0.02
Ethanol
0–1
0.981
0.60  0.03
a
Standard deviation obtained by fitting Equation 4.4 to the data plotted in Figure 4.7.
4.5.4 Effect of Isotope Labeled Alcohols
Additional evidence for an interaction between the alcohols and the mixed valence cluster
could possible be acquired by using spin labeled alcohols. Regular alcohols consists
mainly of 1H, 12C, and 16O isotopes, where carbon and oxygen both have nuclear spin I =
0 and hydrogen I = ½. By using deuterium (D, I = 1) and 13C (I = ½) labeled alcohols,
additional perturbations of the EPR spectra due to coupling between the spin active
isotope atoms and the diiron-oxygen cluster would further indicate an interaction between
the alcohols and the mouse R2 mixed valence cluster. Unfortunately no changes were
observed when CD3CD2OD, CD3OD, and 13CH3OH were added to the samples. The
distance between the spin active nuclei and the mouse R2 mixed valence cluster may
have been to long for a signal distortion to be resolved.
4.5.5 Microwave Powersaturation Behavior of the Novel EPR Signals
Structural changes around the spin center of interest can be probed by progressive
microwave saturation studies. As mentioned in Chapter 3.9.6, the spin relaxation times T1
and T2 represent spin-lattice and spin-spin relaxation pathways, respectively. Distortions
of the ligand structure around a paramagnetic cluster due to complexation might open
new lattice relaxation pathways, resulting in altered power saturation behavior compared
to the native, undisturbed structure.
The double integral of the EPR absorption first derivative spectrum is proportional to the
square root of the applied microwave power when unsaturated. When the applied
52
Chapter 4
Results and Analysis
microwave power is high enough and the EPR signal becomes saturated and loose
intensity, the microwave absorption rate has become faster than the relaxation rate.
Progressive microwave saturation at a fixed temperature is used to determine the halfsaturation point (P1/2) of a paramagnetic species. Half-saturation values of several species
obtained at the same temperature can be directly compared, and differences in relaxation
properties can be discovered. An empirical equation that are readily used to determine
P1/2 values is
 EPR first derivative

KP1/2
P 
1   1/2 
 P 
Eq. 4.5
b
2
where K is a scaling factor, P the variable microwave power, and b the inhomogeneity
parameter.97 The b factor is fixed to 1 or 3 when the dominating lineshape is Gaussian
(spin-spin and spin lattice relaxation) or Lorentzian (spin-lattice relaxation), respectively.
An intermediate value of b (3  b  1) is allowed when a mixture of lineshapes is
observed.
Values of b below 1 have no physical relevance in the model presented above. However,
(Double Integral /P )/ (Double Integral /P )0
1
1
B
1/2
1/2
A
1/2
1/2
(Double Integral /P )/ (Double Integral /P )0
it is considered a signature of paramagnetic clusters that relaxes through a dipolar
0.1
1
10
0.1
100
1
10
100
Power (mW)
Power (mW)
Figure 4.8 Progressive power saturation of the native (A) and the ethanol-perturbed (B) mixed valence
signals. A; solid line; P1/2 = 11.1  2.2 mW, b= 0.77  0.06, dotted line; P1/2 = 19.6  1.2 mW, b=1.0. B; P1/2
= 16.4  2.4, b= 0.99  0.07. Both data sets were recorded at 4.4 K, and double integrals of EPR first
derivatives were compared with a 1 mM Cu(ClO4)2 standard.
53
Chapter 4
Results and Analysis
exchange coupling with a nearby paramagnetic species.98
The native and the ethanol-perturbed mixed valence clusters were characterized by
progressive power saturation. Equation 4.5 was used to analyze the data (Figure 4.8).
As illustrated in Figure 4.8 A, the best fit for the native mixed valence data was obtained
using Equation 4.5 with b = 0.77  0.06 and P1/2 = 11.1  2.2 mW (solid line). This
indicates the presence of a weak dipolar exchange coupling between the mixed valence
cluster and a nearby paramagnetic center not being a radical.99 . In Figure 4.8 A, it can be
noticed that the difference between the solid line (b = 0.77, P1/2 = 11.2) and the dotted
line (b = 1, P1/2 = 19.6) is not very large. Thus, it is not appropriate to speculate whether
there is an dipolar exchange coupling present between the mixed valence cluster and a
nearby paramagnetic cluster or not without additional experimental data. Analysis of the
progressive microwave saturation of the ethanol-perturbed EPR yielded P1/2 = 16.4  2.4
mW and b = 0.99  0.07 (Figure 4.8 B).
The methanol-perturbed EPR signal was
also studied by progressive power
1/2
(Double Integral /P )/ (Double Integral /P )0
saturation (Figure 4.9). When the data was
fitted using Equation 4.5, the b value was
fixed to 1 and P1/2 was allowed to float in
1/2
order to obtain comparable P1/2 values.
1
The two P1/2 values were comparable in
magnitude; 22.8  1.0 mW and 28.6  2.3
mW for the native and methanol-perturbed
1
EPR signals, respectively. This indicates
10
100
Power (mW)
that the relaxation properties of the mixed
Figure 4.9 Progressive powersaturation of native
(solid line) and methanol-perturbed (dotted line)
mixed valence signal. Data was fitted with Equation
4.6, without methanol; P1/2 = 22.8  1.0 mW, with
methanol; P1/2 = 28.6  2.3 mW. The b-value was
fixed to 1.Temperature 4.2 K.
valence cluster did not change
significantly in the presence of methanol.
The results from the progressive power
54
Chapter 4
Results and Analysis
saturation studies of the mixed valence cluster are summarized in Table 4.6.
From these results it can be suggested that neither methanol nor ethanol change the
relaxation properties of the mixed valence cluster significantly. Hence, the progressive
powersaturation studies neither verified nor rejected the hypothesis that alcohol can
interact directly with the mouse R2 mixed valence cluster.
Table 4.6 Results from progressive microwave power saturation studies
Species
b- value a
P1/2 (mW) a
Mouse R2 mixed valence
0.77  0.06
11.1  2.2
(T = 4.2 K)c
Mouse R2 mixed valence in 1 M ethanol
0.99  0.07
16.4  2.4
(T = 4.2 K)c
Mouse R2 mixed valence
1b
22.8  1.0
(T = 4.4 K)c
Mouse R2 mixed valence in 1 M methanol
1b
28.6  2.3
(T = 4.4 K)c
a
Standard error given by fitting program. b Few data points were not compatible with a floating b-value
during the fitting procedure, hence it was fixed to 1. c The temperature measuring device have a constant
positive unknown offset. Thus the temperatures tabulated here are those displayed by the measuring device
and not the real ones.
4.6 Theoretical Studies of the Mouse R2 Mixed Valence Cluster
4.6.1 Purpose of the Theoretical Studies
In the preceding chapter, it was shown that the addition of primary alcohols induced
perturbations in the mouse R2 mixed valence EPR spectra. Titration experiments with
methanol and ethanol indicated that alcohol can bind to the mixed valence cluster. It was
of interest to connect the native and alcohol-perturbed EPR signals to the structure of the
diiron-oxygen cluster. To do that, advanced calculations to estimate the spin Hamiltonian
parameters that are related to ligand field theory were performed.
The study described here consists of two elements; simulations of experimental EPR
spectra using a phenomenological spin Hamiltonian and ligand field calculations based
on second order perturbation theory.
55
Chapter 4
Results and Analysis
4.6.2 Simulation of Experimental EPR Spectra
Information regarding the properties of a paramagnetic spin system can be obtained by
simulation of experimental EPR spectra using a phenomenological spin Hamiltonian. The
elements of the spin Hamiltonian for a dinuclear coupled transition metal cluster are
briefly explained in Chapter 3.9. The operator is given as


~
~
ˆ  2JŜ Ŝ  B~

ga Ŝa  B~
g bŜb  Ŝa Da Ŝa  Ŝb D bŜb (Equation 3.14).
a b
The source code for the program ddpowjea100 written in Fortran 77 was kindly provided
by Dr. Joshua Telser, Roosevelt University, Illinois. The program creates an energy
matrix from the input parameters briefly described in Chapter 6.3. A dinuclear Sa = 2, Sb
= 5/2 antiferromagnetic coupled cluster yield a 30x30 energy matrix when all 30 possible
spin transitions are included (calculated from (2Sa+1)*(2Sb+1) = 30). Matrix
diagonalization yields transition energies (eigenvalues of the matrix) and transition
probabilities (eigenvectors of the matrix) that are combined with linewidth functions to
generate a theoretical EPR spectrum. Initial assumptions applied when calculating spin
Hamiltonian values for diiron-oxygen mixed valence clusters with low isotropic
exchange coupling (-30 cm-1 < J) are:
1. The ferric g-tensor (gx, gy, gz) is set to (2.0, 2.0, 2.0) due to the 6S ground state of the
ferric iron.2,101
2. Zero field splitting values D and E of the non heme ferric iron is very small compared
to those of the non heme ferrous iron, and a value of |D| < 1 cm-1 can be
assumed.2,60,102
When calculating spin Hamiltonian parameters of mixed valence or diferrous diironoxygen clusters from EPR, Mössbauer, and MCD data, two relationships between the
ferrous zero field splitting values (D and E), the ferrous g-tensor(s) and the spin orbit
coupling ( = -100 cm-1) are usually employed:2,3,60,101,103,104
56
Chapter 4
D
E
Results and Analysis
2g z  g x  g y 
Eq. 4.6
4
 (g x  g y )
Eq. 4.7
4
Equation 4.6 and 4.7 are only valid when the 1/3  E/D  0.
When applying the assumptions regarding the ferric iron and the relationships between
the ferrous g-tensor and zero field splitting values (Equation 4.6 and 4.7), the number of
spin Hamiltonian parameters that could be varied were reduced considerably. The
parameters varied during the simulations were: the z-component of the ferrous iron gtensor gz, the ferrous iron axial zero field parameter D, the ratio between the ferrous iron
rhombic and axial zero field splitting parameters E/D, and the isotropic exchange
coupling constant J.
General trends were noticed:
1. The gz value controlled the position of the observed effective g-value, gxeff.
2. The D and E values mainly determined the positions of gyeff and gzeff.
3. The ratio of D/J determined the broadness of the spectra. The range of possible J
values were estimated to -30 cm-1 < J < -10 cm-1 by considering the spectra in Figure
4.10 and 4.11 of the limit 5 cm-1 < D < 8 cm-1. Larger values of D are in principle
possible. However, they are excluded by the fact that J for the mouse R2 mixed
valence cluster are not lower than the value found for the mixed valence methane
monooxygenase hydroxylase (J = -30 cm-1)2 that has a narrower EPR signal (see
Figure 2.9). Estimated D-values of ferrous irons are usually larger than 5 cm-1.
The isotropic exchange constant J for the mouse R2 mixed valence cluster was assumed
ˆ  2JŜ Ŝ ). This assumption was necessary, and is built on the
to be -16.4 cm-1 ( 
ex
a b
experimental values estimated for uteroferrin. The observed geff values (1.93, 1,74, 1.59)
for uteroferrin are similar to those observed for the mouse R2 mixed valence cluster (see
Figure 2.9). Uteroferrin, a purple acid phosphatase isolated from pig allantoic fluid, has a
mixed valence Fe(II)-Fe(III) active form. The diiron-oxygen cluster in uteroferrin has
been characterized by EPR,58 Mössbauer,59,61,65 and MCD60 spectroscopy.
57
Chapter 4
Results and Analysis
Different values of J, D and E/D for this cluster have been estimated (Table 4.7), and
significant deviations between the determined values are apparent. EPR spectra simulated
using plausible parameters are compared to geff values observed for the native mixed
valence mouse R2 EPR signals in Figure 4.10 and 4.11. From those illustrations it can be
suggested that the J value for the mouse mixed valence cluster is in the range –30 cm-1 <
J < -10 cm-1. Given the range of published J-values for uteroferrin, an analysis of the
mouse mixed valence EPR spectra with J = -16.5 cm-1 is not unreasonable.
eff
g =1.73
eff
-1
EPR FIRST DERIVATIVE
J (cm )
-5
-10
-20
-30
-40
-50
g =1.92
eff
g =1.73
-1
D (cm )
EPR FIRST DERIVATIVE
eff
g =1.92
11
10
9
8
7
6
3600
4000
3600
4400
4000
4400
Field (G)
Field (G)
Figure 4.10 Simulated EPR spectra where the
ferrous ZFS parameters D and E/D were fixed at 5
cm-1 and 0.2, respectively. The isotropic exchange
parameter was varied from –5 to -50 cm-1. By
considering the position of the gxeff =1.92 and gyeff=
1.73 of mouse R2 mixed valence in respect to the
simulated spectra, only –30 <J<-10 makes sense.
Figure 4.11 Simulated EPR spectra where E/D and J
were fixed to 0.2 and –30 cm-1, respectively. The
ferrous D value was varied from 6-11 cm-1. In the
limit –30 cm-1 < J, the possible D values are found
between approximately 6 and 8 cm-1.
Table 4.7 Spin Hamiltonian parameters for uteroferrin
Reference
Technique
ˆ  2JŜ Ŝ )
J (cm-1) ( 
ex
a b
D (cm-1)
E/D
Yang et al.60
MCD
< -5
5.17
0.17
Antanaitis et al. 58
EPR
-7
-
-
Mössbauer
-10
-8
0.18
Mössbauer
-17.3
+10.8
0.29
Sage et al.
65
Rodriguez et al. 61
58
Chapter 4
Results and Analysis
The simulated (SIM) spectrum of the
experimental native mixed valence EPR
signal (EXP) is shown in Figure 4.12 A.
A
Using a Gaussian lineshape, a good
SIM
simulation was obtained with the parameters
listed in Table 4.8. For the alcohol perturbed
of both Gaussian and Lorentzian elements,
and the intensities of the first derivative
peaks were not possible to reproduce.
However, the spin Hamiltonian parameters
are obtained when the effective g-values of
the experimental and simulated spectra are
EPR FIRST DERIVATIVE
EPR signals, the lineshape seemed to consist
EXP
B
SIM
EXP
C
the same, regardless of the intensity of the
SIM
spectral peaks. The same J value was kept
EXP
during all simulations so that the g-tensors
and zero field splitting parameters could be
compared for all mixed valence species.
3600
4000
Simulated spectra of methanol and ethanol
4400
Field (G)
perturbed EPR spectra are shown in Figure
Figure 4.12 Simulated (SIM) and
experimental (EXP) EPR spectra of mouse R2
mixed valence cluster with A; nothing added,
B; 1 M methanol, C; 1 M ethanol.
Experimental spectra were recorded at 4 K, 5
mW microwave power, and 9.65 GHz.
4.12 B and C, respectively. The spin
Hamiltonian parameters for the mixed
valence cluster interacting with methanol or
ethanol are listed in Table 4.8.
Table 4.8 Spin Hamiltonian parameters for the mouse R2 mixed valence cluster
Mixed valence species
Fe(II) g-tensor (x,y,z)
J (cm-1)
Fe(II) D (cm-1)
E/D
Nativ
(2.167, 2.222, 2.075)
-16.5
5.97
0.23
+ Methanol
(2.154, 2.198, 2.052)
-16.5
6.20
0.175
+ Ethanol
(2.150, 2.193, 2.052)
-16.5
5.97
0.18
59
Chapter 4
Results and Analysis
4.6.3 Ligand Field Calculations
The zero field splitting D and E spin Hamiltonian parameters obtained for the mouse R2
mixed valence species in Chapter 4.6.2 were related to ligand field theory through second
order perturbation theory (Equation 4.6 and 4.7).
Thus, the coordinating environment of the ferrous iron in the Fe(II)-Fe(III) cluster can be
estimated provided that second order theory is valid for these systems.
Since the magnitude of the isotropic exchange coupling between the ferric and the ferrous
irons initially was guessed, the axial zero field splitting parameter D for the ferrous iron
determined by spin Hamiltonian simulations contain errors.
The main purpose of the spin Hamiltonian calculations and ligand field calculations was
to estimate the changes of the ligand field of the ferrous iron that could occur when an
alcohol interacted with the mixed valence cluster.
In a free ferrous iron the d-orbitals are
degenerate, meaning they posses the same
energy (see Figure 4.13). When a ferrous iron
is octahedrally coordinated (Oh) by ligands
donating electrons to the 4s, 4p, 3d(z2) and
3d(x2-y2) bonding orbitals, the energy levels
of the antibonding 3d(z2)* and 3d(x2-y2)*
orbitals are separated from the non-bonding
3d(xy), 3d(xz), and 3d(yz) orbitals by 10Dq
(about 12000 cm-1). The non-bonding d(xy),
d(xz) and d(yz) orbitals, termed the 5T2g
Figure 4.13 Splitting of the ferrous iron dorbitals. Pure octahedral coordination (Oh) split
the orbitals into the 5Eg and 5T2g set while axial
and rhombic distortion results in further splitting.
orbital set, are lower in energy compared to
the antibonding 5Eg orbital set consisting of
3d(z2)* and 3d(x2-y2)*. Since Fe(II) is a d6
atom, 6 electrons are distributed among the 5 d-orbitals of the 5Eg and 5T2g sets. The 5Eg
and 5T2g sets contain 2 and 4 electrons, respectively, when the energy splitting between
5
Eg and 5T2g is smaller than the electron pairing energy. Such an electron distribution
60
Chapter 4
Results and Analysis
results in a total spin S =2 and is referred to as the high spin state of Fe(II). When the
ferrous iron experience interactions along the z-axis, the energy of the d(z2)*, d(xz), d(x2y2)*, and d(yz) orbitals increase compared to the d(xy) orbital that is less affected by this
axial distortion. The energy difference between the doubly degenerated d(xz), d(yz)
orbital set and d(xy) are given by . If the direction of the axial distortion is tilted with
respect to the z-axis, the d(xz) and d(yz) orbitals will be separated by an energy V. Thus
the ferrous iron experience a rhombic ligand field.
Approximate energies of d-orbitals split by octahedral and distorted octahedral ligand
fields are given in Figure 4.14. Figure 4.13 and 4.14 were taken (and modified) from
Solomon et al.105
Figure 4.14 Ligand field splittings of the d-orbitals according to A;
pure octahedral, B; strong axial and C; weak axial coordination.
61
Chapter 4
Results and Analysis
The values of the calculated spin Hamiltonian parameters presented in Chapter 4.6.2 are
directly connected to ligand field theory for strong axial distortion of a octahedral
coordinated complex by the following equations:2,67,106
E( xz ) 
g e
gx  ge
Eq. 4.8
E( yz ) 
g e
g y  ge
Eq. 4.9
The energies E(xz) and E(yz) are given with respect to the ground state orbital d(xy),
which has the energy E(xy) = 0. In all three equations above, the many electron spin orbit
coupling constant  is 100 cm-1 (only for those equations), ge is the free electron g-value
and gx, gy, and gz are the elements of the ferrous iron g-tensor.
The expressions for the 5T2g zero field parameters in Figure 4.13 are then given by
E ( xz )  E( yz )
2
Eq. 4.10
V  E( xz )  E( yz )
Eq. 4.11

All energies obtained from the ligand field calculations using Equations 4.8-11 are
summarized in Table 4.9, and the splitting of the 5T2g set is illustrated in Figure 4.15.
Table 4.9 Ligand field energies of the ferrous iron in the mixed valence clustera
Mixed valence species
E(yz) (cm-1)
E(xz) (cm-1)
 (cm-1)
V (cm-1)
Nativ
912
1216
1064
304
+ Methanol
1024
1317
1170
293
+ Ethanol
1051
1357
1204
306
a
All energies are given in respect to the d(xy) ground state.
62
Chapter 4
Results and Analysis
1500
xz
xz
yz
yz
-1
Energy (cm )
xz
1000
yz

500
0
V
xy
Nativ mixed
valence
xy
xy
+Methanol
+Ethanol
Figure 4.15 Ligand field splittings of the T2g set of the ferrous iron in
the mouse R2 mixed valence cluster. An increase in  are observed
when alcohol was added while the values of V did not change much.
4.6.3 Summary of Results From Theoretical Calculations
Due to uncertainty in the exchange coupling constant J used throughout the spin
Hamiltonian calculations, the zero field parameters and thus ferrous g-tensors obtained
contain errors. These errors consequently affect the results from the ligand field
calculations. However, the theoretical calculations were performed to evaluate possible
scenarios for the alcohol-mixed valence cluster interaction that could be compared to
very similar results from studies of other mixed valence diiron-oxygen proteins.
With this perspective the theoretical calculations were successful. A further analysis is
presented in the discussion.
63
Chapter 4
Results and Analysis
4.7 CD and Light Absorption Studies of the Mouse R2 Diferric Cluster
4.7.1 Purpose of the Experiments
The exciting observation that alcohols interacted with the mixed valence cluster in mouse
R2 encouraged us to investigate whether the alcohols interacted with the active diferric
form of mouse R2. Since EPR mainly detects half integer spins, a different method had to
be used for this purpose. For reconstituted mouse R2, charge transfer transitions between
the ferric irons and the oxygen bridge can be observed in the 300-450 nm region (based
on observations for E.coli R2).69,107 In addition, a band originating from a vibronic
progression of a ~1500 cm-1 C-O vibration of the unprotonated tyrosyl radical appear at
416 nm.107 An interaction between methanol and the active R2 diferric cluster could
possible be probed by CD and light absorption spectroscopy, thus spectra were recorded
in the presence and the absence of methanol.
4.7.2 Reconstituted Mouse R2 in the Presence of Methanol
mouse R2 with methanol in the range
from 50 mM to 1 M gave no changes
in the CD spectrum (Figure 4.16).
However, the CD spectra were
recorded after only 1 minute
Relative ellipticity
A titration of 160 M reconstituted
incubation with methanol, and the CD
300
bands between 290 and 320 nm were
not possible to detected due to
interference from the protein in that
region.
350
400
450
Wavelength (nm)
Figure 4.16 CD spectra of 160 M
reconstituted mouse R2; solid line; nothing
added, dotted line; 1 M methanol added. No
difference in peak positions or signal intensity
was observed. Spectra were recorded at 4 C.
64
Chapter 4
Results and Analysis
When studying the diferric cluster by light absorption spectroscopy, the difference
spectra were recorded in order to eliminate all spectral contributions from the protein.
The sample was incubated in the presence of 0.5 M methanol in a sealed cuvette for about
12 hours at room temperature. In Figure 4.17, a difference can be noticed in the spectra of
reconstituted mouse R2 in the presence and the absence of methanol. Remarkably, the
signal seems to be unchanged in the low wavelength region while a decrease in intensity
is observed in the higher wavelengths.
Two explanations for these observations can be suggested:
1. Some of the iron-oxo charge transitions and tyrosyl radical bands are influenced by
addition of alcohol.
2. The long incubation time at room temperature resulted in disassembly of the diironoxygen cluster.
The former explanation is supported by the
2.0
Absorbance
observation that the iron-oxo charge
transfer band at ~315 nm seems unchanged
after the long incubation with methanol,
indicating that the diiron-oxygen cluster is
1.5
1.0
0.5
intact. However, the experiment has not
been repeated.
300
350
400
450
500
 (nm)
Thus, the conclusion from the CD and light
Figure 4.17 UV-absorbance difference spectra of
~100 M active mouse R2 (solid line) and the
same sample after 12 hours incubation in presence
of 0.5 M methanol at ~20 C (dotted line).
absorption experiments must be that there
is a possibility that methanol interacts with
the diferric cluster. Further studies should
be undertaken.
65
5 Discussion
5.1 Introduction
The work presented in this thesis has mainly been focused on the redox and spectroscopic
properties of the mixed valence redox state of the diiron-oxygen cluster in mouse R2.
Introductory studies such as R2 reconstitution and redox studies are considered in the first
two sections of this chapter, while the novel interaction between alcohols and the mixed
valence cluster and possible implications of this finding are discussed in the last sections.
5.2 Tyrosyl Radical Content in Reconstituted Mouse R2
Theoretically, both monomers of the R2 homodimer bind two ferrous irons that
subsequently react with dioxygen to form a diiron-oxygen cluster and a tyrosyl radical.
Consequently, quantification of the tyrosyl radical content should yield 2 radicals per R2
dimer. Yields of 1 – 1.5 radicals have been reported earlier.69,88,89 Our novel approach for
mouse R2 reconstitution yielded a radical content of 1.7  0.1 radicals per R2 dimer. The
obtained high radical yield confirmed that the reconstitution of the diiron-oxygen cluster
and tyrosyl radical in mouse R2 was successful.
5.3 Redox Chemistry of PMS and Mouse R2
The main purpose of the redox studies of the electron transfer mediator PMS and the
diiron-oxygen cluster in mouse R2 was to establish a system where reproducible large
amounts of the mouse R2 mixed valence cluster could be obtained. High yields of the
mixed valence cluster are important when employing techniques such as EPR,
Mössbauer, ENDOR, and MCD spectroscopy. It was also of interest to estimate the
67
Chapter 5
Discussion
midpoint potential for the reduction of the diiron-oxygen cluster in mouse R2 and relate
the obtained values to midpoint potentials determined for other diiron-oxygen proteins.
Earlier attempts to determine the redox potentials of the diiron-oxygen cluster in mouse
R2 by using low concentrations of electron transfer mediators (~0.1 mM) combined with
a voltmeter, electrodes, and EPR have failed (K.K. Andersson, personal communication).
Such an experiment usually requires about 10-20 hours, depending on how many samples
to be made. It has been suggested that the mouse R2 diiron-oxygen cluster disassembles
within that time window (K.K. Andersson, personal communication). Thus it has been
difficult to determine the redox potentials of the diiron-oxygen cluster.
Based on the rapid redox equilibration observed when 5 mM of the electron transfer
mediator PMS was used (see Figure 4.4), we suggest that the experiment mentioned
above should be repeated using such a concentration of PMS. Then disintegration of the
diiron-oxygen cluster during the experiment might be avoided.
The various redox states of the mouse R2 diiron-oxygen cluster can be obtained by
varying the redox potential of the protein solution. A high yield of mixed valence cluster
was obtained by utilizing a redox buffer to adjust the redox potential of the solution (Esol’)
to a value close to the Em’ estimated for the reduction of the mouse R2 diiron-oxygen
cluster.
When studying the redox equilibrium, it was important to understand the properties of the
redox buffer determining the redox potential of the solution. Oxidized and reduced PMS
constituted the redox buffer, and Esol’ was adjusted by adding the potent reductant DT.
The Esol’ values were calculated from the Nernst equation (Equation 4.1) on the basis of
the midpoint potentials previously determined for the PMS+/PMSH redox couple. Thus, it
was important to find out whether the PMS+/PMSH redox couple was formed in a
predictable fashion when DT was added or not.
In a previous study of PMS, nicotine amide dinucleotide (NADH) was used to reduce
PMS+.86 Addition of equimolar amounts of NADH to a PMS solution did not result in
complete reduction of PMS+ and a complex formation between PMS+ and PMSH was
68
Chapter 5
Discussion
suggested.86 When investigating the reduction of PMS by DT, we found a linear
dependence of the reduction of PMS+ to PMSH in respect to the concentration of DT.
Non of our observations confirmed a presence of a (PMSHPMS)+ complex. Thus, we
suggest that that the [PMS+]/ [PMSH] ratio is formed in a predictable fashion in the
presence of DT, and that the redox potential of a PMS/ DT solution can be estimated by
using the Nernst equation.
Samples containing reconstituted mouse R2, PMS, and DT were prepared and the yield of
mixed valence cluster was quantified by double integration of the EPR absorption first
derivative spectra. Esol’ values were calculated for each sample using the Nernst equation.
Three models, differing in the number of electrons involved in the electron transfers,
were used to explain the data. This gave Em’ values between 52 and 62 mV (versus the
SHE), which are within previous estimates where –70 mV < Em’ < 80 mV.108 With E.coli
R2, both ferric irons are reduced simultaneously in the presence of DT and electron
transfer mediators. The redox potential for this reduction has been determined to be –115
mV versus the SHE.38 Thus, the estimated midpoint potential for mouse R2 is more
similar to the one determined for MMOH (OB3b), which is 48 mV (versus the SHE).1
The regulatory protein, termed component B, of the MMO complex has been suggested
to alter the structure of the diiron-oxygen cluster containing MMOH protein.109 The
midpoint potential for the diiron-oxygen cluster in MMOH (OB3b) is shifted to –84 mV
(versus the SHE) in the presence of component B.1 It would be of interest to estimate the
midpoint potential for the diiron-oxygen cluster in mouse R2 in the presence and absence
of alcohol to pursue a possible structural effect of alcohol on the metal cluster.
5.4 Small Alcohols might Bind to the Mouse R2 Mixed Valence Cluster
Both MMOH (Bath and OB3b) and mouse R2 contain analogous diiron-oxygen clusters
that can have diferric Fe(III)-Fe(III), mixed valence Fe(II)-Fe(III) and diferrous Fe(II)Fe(II) redox states. A common approach when investigating structural properties of these
69
Chapter 5
Discussion
clusters is to probe the ability of exogenous ligands to interact with the diiron-oxygen
cluster in several oxidation states.
The scientific relevance of these studies can be justified by:
1. The dissociation of hydroxylated substrate from the diiron-oxygen cluster is the ratelimiting step in the MMO catalytic cycle.51 Thus, the productdiiron-oxygen cluster
complex (compound T) is an important intermediate in this cycle. Compound T and
similar alcoholdiiron-oxygen cluster complexes have been characterized by
spectroscopy2,53,54 and crystallography.52 Interestingly, our results indicate that the
nature of the interaction between alcohols and the mouse R2 mixed valence cluster
might resemble compound T. This is fascinating in respect to the unrelated functions
of the diiron-oxygen clusters in mouse R2 and MMOH.
2. The ability of small molecules to interact with the diiron-oxygen cluster in R2 is also
of medical interest. When constructing R2 diiron-oxygen cluster specific inhibitors,
important elements to consider are the nature of the interaction and the affinity of the
inhibitors towards the diiron-oxygen cluster. Data on how small molecules interact
with the diiron-oxygen cluster form a basis for understanding these complex
interactions.
Our hypothesis, based on the titration experiments and the theoretical calculations, is that
primary alcohols can bind to the mixed valence cluster in mouse R2. By comparing our
results to information achieved from studies of MMOH, we can suggest plausible models
to explain our observations.
Previous studies of the diiron-oxygen clusters in MMOH and R2 have revealed that the
affinity of various exogenous ligands to the diiron-oxygen cluster depends on the redox
states of the two irons. The diferrous cluster in MMOH (OB3b), in which both irons are
5-coordinated,47,110 does not bind any anionic ligands (e.g. azide) or enzymatic products
tested.110 In E.coli and mouse R2, however, azide bind to the diferrous cluster.25,37,111
70
Chapter 5
Discussion
The mixed valence cluster of MMOH (Bath) binds methanol to the ferrous iron,54 but no
binding of anionic ligands to this cluster have been reported. The presence of 500 mM
azide does not alter the mouse R2 mixed valence cluster (K.K. Andersson, personal
communication). Results presented in this thesis indicate that primary alcohols can.
Recent crystallographic studies of MMOH (Bath) have shown that methanol and ethanol
bind in a specific manner between the two ferric irons in the oxidized diiron-oxygen
cluster.52 One might speculate that methanol interacts with the diferric cluster in mouse
R2 on the basis of the light absorption study presented in Chapter 4.7. Further
experiments are required to verify or reject this hypothesis.
The binding curves for methanol and ethanol illustrated in Figure 4.9 suggest a specific
interaction between the two alcohols and the mouse R2 mixed valence cluster. Since the
Kb values are 0.24 M and 0.6 M for methanol and ethanol, respectively, it can be argued
that the shifts of the effective g-values originates from a general solvent effect that
disturbs the protein tertiary structure. However, our hypothesis is supported by several
observations:
1. Research focused at protein stability in various solvents indicates that the alcohol
concentrations used in our studies do not denaturate the mouse R2 protein (see Table
5.1).
2. When the binding of methanol and ethanol to the diferric cluster in MMOH was
studied by crystallography, the protein crystals were soaked in 1 M methanol and 0.9
M ethanol, respectively prior to data collection.52 In these two crystal structures, only
slight changes in the iron coordinating environment due to alcohol binding were
observed.
3. The effect of 1 M methanol and 1 M ethanol on the mouse R2 mixed valence EPR
spectrum is more pronounced than for 1 M concentrations of the more denaturing
alcohols 1-propanol and 1-butanol.
Thus, we suggest that the perturbations of the mouse R2 mixed valence EPR signals
recorded in the presence of the selected alcohols originate from an interaction of alcohol
with the diiron-oxygen cluster.
71
Chapter 5
Discussion
Table 5.1 Denaturation midpoints for proteins in organic solvents112
Protein
Myoglobina
Cytochrome c
a
-Chymotrypsinogenb
a
[Methanol] (M)
[Ethanol] (M)
[1-propanol] (M)
[1-butanol] (M)
12.4
5.3
2.0
0.8
12.5
7.4
4.0
-
7.2
3.8
1.6
0.7
pH = 5.7. b pH = 2.8. All values are determined at 25 C.
The weaker affinity of 1-propanol and 1-butanol to the mixed valence cluster can
possibly be explained by their larger size compared to methanol and ethanol. Since the
diiron-oxygen cluster is positioned inside a pocket, insufficient space in the pocket may
lead to exclusion of 1-propanol and 1-butanol.
The theoretical calculations also support our hypothesis that alcohol can bind to the
mixed valence cluster. Analogous calculations have been performed at the alcohol
perturbed mixed valence EPR signals recorded for MMOH (OB3b).2 Simulations of the
native and alcohol perturbed mouse R2 mixed valence EPR spectra resulted in sets of
spin Hamiltonian parameters. Those parameters could be related to the ligand field
energies for the ferrous iron of the cluster when assuming a strong axial interaction.
Ligand field energies calculated from spin Hamiltonian parameters for mouse R2 (this
work) and MMOH (OB3b)2 are compared in Figure 5.1. Only the orbitals belonging to
the T2g set that is described by the zero field parameters  and V are included in that
illustration. As expected, the energies of the native, unperturbed mixed valence clusters in
mouse R2 and MMOH (OB3b) are not similar due to structural differences. Upon
methanol addition to the native MMOH (OB3b) mixed valence cluster, the zero field
splitting parameter  was lowered by 190 cm-1 and the change in V was 5.7 %.2 For
mouse R2, the changes in  were 106 and 140 cm-1 in the presence of methanol and
ethanol, respectively. The relative variations of V were also small for the mouse R2
cluster (3.7 and 0.6 % in the presence of either methanol or ethanol).
The comparable magnitudes of the changes in the ligand field energies upon alcohol
addition observed for the MMOH (OB3b) and mouse R2 mixed valence clusters suggest
that methanol and ethanol can bind to the ferrous iron in the mouse R2 cluster. However,
72
Chapter 5
Discussion
since the theoretical calculations were based on the set of assumptions given in Chapter
4.6.2, the calculated parameter set should only be considered as one of several possible
solutions.
Thus, no certain conclusions can be made based on the ligand field treatment.
MMOH
-1
Energy (cm )
Mouse R2
xz
2000
xz
1000
V
yz


0
V
yz
xy
xy
Nativ +MetOH +EtOH
Nativ +MetOH
Figure 5.1 Calculated ligand field energies for the T 2g orbital set for the ferrous
iron in the mouse R2 and MMOH (OB3b) mixed valence cluster.
Possible binding of methanol and ethanol to the mouse R2 mixed valence cluster can be
modeled by considering data that have been published for E.coli R2 and MMOH because:
1. All amino acids involved in iron binding and the radical transfer pathway are highly
conserved in E.coli and mouse R2.32
2. X-ray structures of mouse32 and E.coli8 R2, show that the positions of the iron
coordinating amino acid sidechains are comparable in both proteins.
3. The coordination and structure of the diiron-oxygen clusters in MMOH and R2 are
very similar. Structures of MMOH (Bath) with methanol or ethanol bound to the
diferric cluster52 might resemble alcohol bound to the mouse R2 mixed valence
cluster.
73
Chapter 5
Discussion
4. Binding of methanol to the ferrous iron of the mixed valence cluster in MMOH
(Bath) have been observed by ENDOR.54 Results from EPR and Mössbauer studies
also support the binding of methanol to the mixed valence cluster in MMOH
(OB3b).42,54
Three plausible modes of alcohol binding to the mouse R2 mixed valence cluster are
illustrated in Figure 5.2. The crystal structures of E.coli R2 and MMOH (Bath and OB3b)
all show that one water is terminally bound to each of the irons in the diferric oxidation
state. In our models it is assumed that the geometry of the iron coordinating environment
in mouse R2 is conserved upon reduction to the mixed valence state, and that an alcohol
replaces the terminally bound water when binding to the cluster.
However, geometric flexibility of the iron coordinating amino acid sidechains have been
shown for both E.coli R2113 and MMOH.47,113 Such a flexibility of the endogenous
ligands of the irons is not included in our models. It is also not known whether it is the
O
A
Asp139
Glu267
H
O
OH2
Asp139
His173
O
O
H 3C
Asp139
O
O
His173
O
N
N
Glu170
O
Fe2
O
Asp139
O
His270
His173
N
O
O
O
Fe1
Glu233
N
Glu267
H
O
O
N
Glu233
O
N
N
O
Fe2
O
H3C
His270
N
O
D
O
Fe2
O
O
Glu170
N
O
O OH2
Fe1
O
His173
Glu267
H
O
O
O
O
N
N
O
C
H
O
His270
N
Glu170
N
OH2
Fe1
Glu233
O
N
O
O
Fe2
Glu267
H3C
O
O
O OH2
Fe1
O
B
O
Glu170
Glu233
N
His270
N
Figure 5.2 Proposed mode of binding of methanol to the mixed valence cluster in
mouse R2. Amino acid sidechains are labeled using mouse R2 amino acid numbering.
A; native mixed valence cluster, B; water replaced by methanol at Fe2, C; water
replaced by methanol at Fe1, C; methanol replaces both waters and forms a bridge
between Fe1 and Fe2.
74
Chapter 5
Discussion
alcohol or alcoxide that might binds to the iron cluster. However, it is not possible to
suggest more precise models due to lack of experimental evidence.
Considering the results from the spin Hamiltonian calculations in Chapter 4.6.2, it is to be
expected that the alcohol interact with the ferrous iron in the mixed valence cluster. Even
though, it is not known which of the irons (Fe1 or Fe2, Figure 5.2) that are reduced. Thus,
Figure 5.2 B is most correct if Fe2 is reduced and Figure 5.2 C when Fe1 is reduced. If the
alcohol binds in a bridging position, Figure 5.2 D is a plausible model. The hydroxide
bridge of the mouse R2 mixed valence diiron-oxygen cluster has been proposed by Atta
et al.37
Similar models to those presented in Figure 5.2 can be used to illustrate possible ethanol
binding to the mixed valence cluster.
5.4.1 Relevance of Results and Further Experiments
The redox experiments show that it is possible to efficiently reduce the diferric form of
mouse R2 cluster to a mixed valence oxidation state by using a high concentration of a
redox buffer. By reproducing the high yield of mixed valence it is now possible to make
samples suitable for Mössbauer spectroscopy.
Interestingly, the midpoint potential Em’ for mouse R2 seems to be close to the one
determined for the diiron-oxygen cluster in MMOH (OB3b). Further experiments,
involving accurately determination of the midpoint potential of the mouse R2 diironoxygen cluster, might verify similarities of the redox behavior of the mouse R2 and the
MMOH diiron-oxygen clusters.
Considering the results obtained from both titration experiments and theoretical
calculations, we suggest that both methanol and ethanol can bind to the mouse R2 mixed
valence cluster. ENDOR, Mössbauer, and MCD spectroscopy are suitable techniques that
can be used to verify this hypothesis. No such experiments have been conducted yet.
75
Chapter 5
Discussion
Those techniques can also be used to confirm whether the diferrous and diferric oxidation
states can bind alcohol or not.
Since the proposed electron transport pathway is suggested to include one of the irons in
the mouse R2 diiron cluster, the mixed valence form might be of physiological relevance.
The presence of alcohol might influence the activity of the R1-R2 holoenzyme. Thus,
studying the RNR enzyme kinetics in the presence of alcohol would be of interest.
Considering the properties of mouse R2 and MMOH that are not shared with E.coli R2, it
might be that the mouse protein is a more appropriate fundament for constructing a
MMOH similar enzyme from R2 than the bacterial protein. Since the major goal of the
Iron-Oxygen Protein Network is to convert R2 to an enzyme capable of substrate
hydroxylation, knowledge of the analogous properties of R2 and MMOH form an
important basis for this work.
76
6 Appendix
6.1 Materials
Chemicals
Source
1-Butanol (> 99.5 %)
1-Propanol (> 99.5 %)
2-Mercaptoethanol
Acetic Acid (100 %, p.a.)
Albumin, Bovine (> 96 %)
Ammonia (25 %, p.a.)
Ammonium Iron(II) sulfate Hexahydrate (p.a.)
Ammonium Sulfate (p.a.)
Argon ((g), 99.9997 %)
Bacto Agar
Bacto Tryptone
Bacto Yeast Extract
Bromphenol Blue
Carbenicillin
Chloramphenicol
Di-Potassium Hydrogen Phosphate
EDTA (> 99 %)
Ethanol (Absolutt Prima)
Ethyl-d5 Alcohol-d (>99 atom % D)
Glycerol (87 %, p.a.)
Helium (lq)
HEPES (> 99.5 %, Free Acid)
Hydrochloric Acid (36 %, p.a.)
Isopropyl -D-Thiogalactopyranoside
Low Molecular Weight Standard
Methanol (>99.5 %, p.a.)
Methyl-13C Alcohol (99 atom % 13C)
Methyl-d3 Alcohol-d (99.8 atom % D)
Nitric Acid (69 %, p.a.)
Nitrogen (lq)
Oxygen (g)
PhastGel Blue R (tablets)
Phenazine Methosulfate (92 %)
Phenylmethylsulfonyl Fluoride (> 99 %)
Potassium Chloride (p.a.)
Potassium Di-Hydrogen Phosphate (p.a.)
Potassium Ferricyanide(III) (>99 %)
Potassium Hydroxide (p.a.)
Sodium Chloride (p.a.)
Merck
Merck
Sigma
Merck
Sigma
Merck
Merck
Merck
AGA
Difco
Difco
Difco
Sigma
Sigma
Sigma
Merck
Sigma
Arcus
Aldrich
Merck
AGA
Sigma
Prolabo
Sigma
Pharmacia
Merck
Aldrich
Aldrich
AppliChem
AGA
AGA
Pharmacia
Sigma
Sigma
Merck
Merck
Aldrich
Merck
Merck
77
Chapter 6
Appendix
Sodium Chloride (p.a.)
Sodium Dithionite (~80 %)
Sodium Dodecyl Sulfate (> 99 %)
Sodium Hydroxide (p.a.)
Streptomycin Sulfate
Toluidine Blue O (80 %)
Tris(hydroxymethyl)aminomethane (> 99.9 %)
Merck
Sigma
Sigma
Merck
Sigma
Sigma
Sigma
Column materials
Source
Diethylaminoethyl Cellulose (DE52)
Sephadex G-25 (medium)
Whatman
Pharmacia
Equipment
Source
Bio-Rad Protein Assay
Butyl Septa (various sizes)
Centricon (YM-50)
Collodion Bags (12000 MWCO)
Cuvettes (quarts)
EPR tubes (707-SQ, 25 cm)
Folding Filter (Ø185mm)
Hamilton Syringes
Millex-GP 0.22 m filter
NAP-5 Columns
Nitrocellulose Filters (0.45 m)
PD-10 Columns
PhastGel (8-25 Gradient)
PhastGel SDS Buffer Strips
Polyethylene tubing (various diameters)
Spin Columns (1 mL)
Bio-Rad
Norton Verneret
Millipore
Sartorius
Teknolab
Wilmad
S&S
Teknolab
Millipore
Pharmacia
Millipore
Pharmacia
Pharmacia
Pharmacia
Becton Dickinson
Bio-Rad
Instruments
Manufacturer
ER 4113 HV Liquid Helium Control System
HP8452A Spectrophotometer
pH meter (420A)
Spectropolarimeter (J-810)
PhastSystem
ESP 300E 10/12 X-band spectrometer
Oxford Instruments
Hewlett Packard
Orion
Jasco
Pharmacia
Bruker
78
Chapter 6
Appendix
Scientific Computer Software
Source
MolMol 2k.1
ddpowjea 4.0
Rasmol 2.6
WinEPR 2.0
Koradi, Reto
Telser, Joshua
Sayle, Roger
Bruker
6.2 The Culture Medium and Buffers
In all mediums and other solutions, Milli Q filtered and ion-exchanged H2O (mqH2O)
was used. Buffer solutions were filtered with a nitrocellulose filter (0.45 m) and
degassed for 20 minutes before they were used.
8 L of LB culture medium:
80 g
80 g
20 g
Bacto Tryptone
Sodium Chloride
Bacto Yeast Extract
Add mqH2O until 8 L. Adjust pH to 7.5 by adding 12 M NaOH. Autocleave at 120 C for
25 minutes. When preparing petri dishes add 15 g Bacto Agar per liter LB culture
medium prior to autoclavation.
Buffers
Stock solution A
1.0 M Phosphate Buffer
pH 7.0
Prepare by mixing a 1.0 M K2HPO4 solution with a 1.0 M KH2PO4 solution until pH =
7.0.
Buffer A
50 mM Tris
1 mM EDTA
pH = 7.5
Adjust pH by adding concentrated HCl to the basic Tris solution.
79
Chapter 6
Appendix
Buffer B
10 mM Phosphate Buffer
1 mM EDTA
30 mM KCl
pH = 7.0
Use stock solution A as a basis for the phosphate buffer.
Buffer C
10 mM Phosphate Buffer
1 mM EDTA
70 mM KCl
pH = 7.0
Use stock solution A as a basis for the phosphate buffer.
Buffer D
50 mM HEPES
100 mM KCl
20 % glycerol
pH=7.5
Adjust pH by adding12 M KOH to the acidic HEPES solution.
PhastSystem Solutions
Phast Loadmix
500 L 10 % SDS solution
100 L 2-Mercaptoethanol
200 L 0.1 % Bromphenol Blue solution
8 L loadmix is added to a 14 L buffered protein sample before boiling.
Phast Staining Solution
mL 0.2 % PhastGel Blue R solution
40 mL 20 % Acetic Acid, 80 % mqH2O
80
Chapter 6
Appendix
Phast Destaining Solution
90 mL Methanol
30 mL Acetic Acid
180 mL mqH2O
Phast preservation solution
8 mL Glycerol
8 mL Acetic Acid
64 mL mqH2O
81
Chapter 6
Appendix
6.3 Input Parameters for the Program ddpowjea
Parameter value
Description
2
2.5
2.167, 2.222, 2.075
5.97
1.373
0
0
0
0
2.0, 2.0, 2.0
N
0.1
0
0
0
0
0
35 b
0
0
0
N
0,0,0
9.6516
0
Y
4
300
80
30
N
1
29
2
30
G
3300
4600
1
G
195,235,145
1000
1
1
1
1
1
1
xxx.txt
xxx.dat
ˆ  JŜ Ŝ .

Spin one (Fe2+)
Spin two (Fe3+)
g- tensor (gx, gy, gz) for spin one (Fe2+)
2nd order axial ZFS parameter (D) for Fe2+ (cm-1)
2nd order rhombic ZFS parameter (E) for Fe2+ (cm-1)
3rd order Zeeman parameter for Fe2+
4th order axial ZFS parameter for Fe2+ (cm-1)
4th order cubic ZFS parameter for Fe2+ (cm-1)
4th order rhombic ZFS parameter for Fe2+ (cm-1)
g- tensor (gx, gy, gz) for spin two (Fe3+)
No rotation of spin one in respect to spin two
2nd order axial ZFS parameter (D) for Fe2+ (cm-1)
2nd order rhombic ZFS parameter (E) for Fe2+ (cm-1)
3rd order Zeeman parameter for Fe2+
4th order axial ZFS parameter for Fe2+ (cm-1)
4th order cubic ZFS parameter for Fe2+ (cm-1)
4th order rhombic ZFS parameter for Fe2+ (cm-1)
Isotropic exchange coupling J (cm-1)
2nd order isotropic exchange coupling J2 (cm-1)
Axial part of dipolar exchange coupling (cm-1)
Rhombic part of dipolar exchange coupling (cm-1)
No rotation of dipolar coupling tensors
Anisotropic exchange coupling Ja (cm-1)
Microwave frequency (GHz)
Perpendicular mode (direction of microwave magnetic component)
Include population weighting of transition intensities
Temperature in K
Number of spectral points
Igloo grid interval (accuracy of integration)
Rho grid interval (accuracy of integration)
No specific intervals for integration (full powder spectra)
Minimum state from which to calculate transitions
Maximum state from which to calculate transitions
Minimum state to which to calculate transitions
Maximum state to which to calculate transitions
Gauss is the unit of the magnetic field
Minimum field (G)
Maximum field (G)
EPR absorption output
Gaussian lineshape
Linewidths in MHz
Linewidth cutoff
Electronic linewidth strain factor for Ms =  2
Electronic linewidth strain factor for Ms =  1
Electronic linewidth strain factor for Ms = 0
Electronic linewidth strain factor for Ms = 5/2
Electronic linewidth strain factor for Ms = 3/2
Electronic linewidth strain factor for Ms = 1/2
Parameter output file
Data output file
ex
a
b
82
Terms and Abbreviations
A


B
B
Bath
CD
Compound P
Compound T
Compound X

D
~
D
DT
E
E1’
E2’
Em’
ENDOR
EPR
Esol’
F
Fe2S2
Ferric
Ferrous
e
~g
ge
geff
Ĥ
HDVV
Ĥ ex
I
Intermediate P
Intermediate Q
J
Kb

LMW
MCD
MMO
MMOH
MMOR
Absorption
The Bohr magneton
Magnetic field vector
Magnetic field
Indicates Methylococcus capsulatus
Circular dichroism
Peroxo Fe(III)-Fe(III) form of the metal cluster in mouse R2
Product Fe(III)-Fe(III) cluster complex in MMOH
Fe(IV)-Fe(III) oxidation state of the metal cluster in mouse R2
Energy splitting of the 5Eg and 5T2g orbital sets
Axial zero field splitting parameter
Zero field splitting tensor
Sodium dithionite
Rhombic zero field splitting parameter
First formal redox potential of a reaction
Second formal redox potential of a reaction
Midpoint potential for a redox reaction
Electron nuclear double resonance
Electron paramagnetic resonance
Redox potential of the solution
Faraday constant
Disulfur bridged diiron cluster
Fe(III) oxidation state of iron
Fe(II) oxidation state of iron
Electron gyromagnetic ratio
Electron g-tensor
Free electron g-value
Effective (observed) g-values
Spin Hamiltonian operator
Heisenberg, Dirac, and Van Vleck
HDVV exchange operator
Nuclear spin
Peroxo Fe(III)-Fe(III) form of the metal cluster in MMOH
Fe(IV)-Fe(IV) oxidation state of the metal cluster in MMOH
Exchange coupling constant
Binding constant
Many electron spin orbit coupling constant
Low molecular weight standard
Magnetic circular dichroism
Methane monooxygenase
Methane monooxygenase hydroxylase
Methane monooxygenase reductase
83
Terms and Abbreviations
Mox
mqH2O
Mred
̂
MWCO

n
NADH
NADPH
OB3b
P1/2
PDB
PMS
PMS+
PMSF
PMSH
R
R1
R2
RNR
rpm
Ŝ
S
SDS-PAGE
SHE
T1
T2
T
TB
UV/ vis
V
ZFS
Oxidized electron transfer mediator
Milli-Q filtered and ion-exchanged water
Reduced electron transfer mediator
Magnetic moment operator of an electron
Molecular weight cutoff
Frequency
Number of electrons involved in a redox reaction
Nicotinamide-adenine dinucleotide
Nicotinamide-adenine dinucleotide phosphate
Indicates Methylosinus trichosporium
Half saturation point
Protein data bank
Phenazine methosulfate
Oxidized PMS
Phenylmethylsulfonyl fluoride
Reduced PMS
Gas constant
Ribonucleotide reductase R1 homodimer
Ribonucleotide reductase R2 homodimer
Ribonucleotide reductase
Revolutions per minute
Electron spin operator
Spin angular momentum quantum number
Sodium dodecyl sulfate polyacrylamide gel electrophoresis
Standard hydrogen electrode
Spin lattice relaxation time
Spin-spin relaxation time
Temperature
Toluidine Blue O
Ultraviolet/ visible light absorption spectrophotometry
Energy of the rhombic splitting of the 5T2g orbital set
Zero field splitting
Standard Amino acids
Alanine
Arginine
Asparagine
Aspartic Acid
Cysteine
Glutamine
Glutamic Acid
Glycine
Histidine
Isoleucine
Ala
Arg
Asn
Asp
Cys
Gln
Glu
Gly
His
Ile
A
R
N
D
C
Q
E
G
H
I
Leucine
Lysine
Methionine
Phenylalanine
Proline
Serine
Threonine
Tryptophan
Tyrosine
Valine
84
Leu
Lys
Met
Phe
Pro
Ser
Thr
Trp
Tyr
Val
L
K
M
F
P
S
T
W
Y
V
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