Introduction
Our work is aimed at making hybrid myoglobins to use in
photochemical studies of catalysis by heme proteins. Although
myoglobin does not perform enzymatic functions in vivo, catalytically
active analogues can be synthesized via the modification of the heme
group (porphyrin) with a photoactive ruthenium bpy pendant arm.
Reconstitution of these into apomyoglobin serves as a model for the
catalytically active heme proteins.
Myoglobin
Mb active site
Synthesis of Bpy Pendant Arm
O
(CH2)7Br
LDA, Br(CH)6Br
•Using this
method, we can
systematically vary
the length and composition
of the pendant arm.
N
(CH2)7 N
O
THF
O
DMF
85%
N
O
KN
87%
N
N
N
N
Conc. HCl
Reflux, 10 hours
(CH2)7NH2
(CH2)NH2
50% NaOH/sec-BuOH
Free base
84%
N
N
N
N
Synthesis of Protoporphyrin IX Derivative
NH
N
CO 2Na
PCl5, EtOH
THF
N
HN
NH
sep. on SiO 2
35%
CO2Na
N
CO2Et
•This protects one
proprionate which will be
nessasary to help stabilize our
reconstituted protein.
N
HN
CO2H
The Amide
Coupling
Reaction
NH
N
N
(CH2)NH2
DECP, (Et)3N
THF
Reflux 48 hours
+
HN
NH
60%
N
N
N
HN
N
N
CO2 Et
CO2H
CO2 Et
Using UV-visible
spectroscopy, we can
determine when the
C7PP or RuC7PP has been
synthesized.
CO NH(C H2)7
C7PP
Base
Hydrolysis
NH
N
N
HN
N
sep. on Al 2O3
Ru (bpy )2Cl2
CO2 H
1N NaOH
THF
CONH(C H2)7
Ru(bpy) 2CO 3
MeOH, H2O
75%
N
1.8
400 nm
1.6
RuC7PP
1.4
1
288 nm
RuC7PP
C7PP
0.8
0.6
0.4
0.2
Wavelength (nm)
600
550
500
450
400
350
300
0
250
Absorbance
1.2
For the C7PP, there should be a 1 to 5 ratio between the
peaks at 288 and 400 nm. The peak at 288 nm
corresponds to bipyridine, and the one at 400 nm
represents porphyrin. The spectrum of RuC7PP will
have an increase for the 288 nm peak, increasing the
aforementioned ratio to 3 to 5, as seen.
N
Metallation of Heme Cofactor
NH
FeCl 2
DMF
Reflux, 4 hours
N
sep. on LH20
N
85%
N
N
Fe
HN
N Cl N
N
N
Ru(bpy)2Cl2
N
CONH(CH2)7
CO2H
Ru(bpy) 2Cl2
CO2H
CONH(CH2)7
N
RuC7FePP
1.2
400 nm
RuC7FePP
1
288 nm
0.6
550 nm
0.4
650 nm
0.2
Wavelength (nm)
75
0
70
0
65
0
60
0
55
0
50
0
45
0
40
0
35
0
30
0
0
25
0
Absorbanc
e
0.8
•A UV-visible spectrum was taken to
verify the incorporation of iron into the
porphyrin of RuC7PP. On the spectrum to
the left, it can be seen that there are only
two Q bands. This is diagnostic of the
metallated porphyrin. The two Q bands
are peaks at 550 and 650 nm.
Unmetallated porphyrins, eg. RuC7PP and
C7PP, have four Q bands.
Preparation of Apomyoglobin
• The synthesis of apomyoglobin occurs in a three step process:
1. Extraction of Native Heme
2. Dialysis
3. Lyophilization
4oC
Myoglobin
Apomyoglobin
The reconstitution of the altered heme is done by mixing it with the
apoprotein, and purifying the reformed holoenzyme by chromatography
Molecular Model
of RuC7FePP
myoglobin
Molecular Model of Hybrid Mb
Characterization of the hybrid
by UV Spectroscopy
•When reconstituted into apomyoglobin, the UV-visible
spectrum resembles what one would expect from a
coupling of Ru(bpy)32+ and FeIII myoglobin. The soret
shifts from 400 nm to 409 nm when reconstituted into
protein.
0.8
Visible spectra of the hybrid Myoglobin
0.6
RuC7Mb
Mb
Ru(bpy)32+
0.4
ca. 4 uM
0.2
0
300
400
500
600
700
Wavelength (nm)
OD
Nanosecond Transient Absorption Spectroscopy
Time
sample
Monochromator
Beamsplitter
Termination
Probe Light
LASER
Photoinduced nanosecond transient absorption
FeIII Soret @
410 nm
FeII Soret @
430 nm
0
1
2
3
Time (us)
4
0
1
2
3
Time (us)
4
•Using nanosecond transient
absorption measurements of
the heme soret bands in the
hybrid Mb we can follow the fast
electron transfer between Rubpy
and the FeMb.
•The transient traces illustrate the back electron transfer, kbet, regenerating
the FeIII/Ru2+ state is 2 x 107 sec-1
Fe III *Ru 2+
h
Fe III Ru 2+
kfet
k fet
Fe II Ru 3+
kbet
hv
Fe III
Ru 2+ (byp) 3
Myoglobin
k - b et
Future Work
his 52
During the next year we will attempt to reconstitute
the RuC7FePP heme cofactor into CcP. In myoglobin
the active site is located on the edge of the protein
whereas in CcP one of the propionate groups is
blocked and the other is recessed from the periphery
of the protein. Due to the topographic differences in
the active sites, it is probable that we will need to
alter the length and characteristics of the pendant
arm in order for successful reconstitution.
trp 51
his 175
CcP Active Site
Other Possible Pendant Groups:
-Re(bpy)(CO)3L
(L= Cl-, Br-, pyridine)
-Methyl viologen
N
NR'
trp 191
Acknowledgments
• Farmer Group
• Greg Qushair
• David Khandabi
• Phuong Do