Structure of coenzyme F420H2 oxidase (FprA), a di-iron
flavoprotein from methanogenic Archaea catalyzing the
reduction of O2 to H2O
Henning Seedorf1, Christoph H. Hagemeier1, Seigo Shima1, Rudolf K. Thauer1,
Eberhard Warkentin2 and Ulrich Ermler2
1 Max Planck Institute for Terrestrial Microbiology, Marburg, Germany
2 Max Planck Institute for Biophysics, Frankfurt am Main, Germany
Keywords
coenzyme F420; crystal structure; di-iron
center; F420H2 oxidase; O2 detoxification
Correspondence
U. Ermler, Max Planck Institute for
Biophysics, Max-von-Laue-Str. 3, D-60438
Frankfurt am Main, Germany
Fax: +49 69 63031002
Tel: +49 69 63031054
E-mail: ulrich.ermler@mpibp-frankfurt.mpg.de
(Received 14 November 2006, revised 11
January 2007, accepted 17 January 2007)
doi:10.1111/j.1742-4658.2007.05706.x
The di-iron flavoprotein F420H2 oxidase found in methanogenic Archaea
catalyzes the four-electron reduction of O2 to 2H2O with 2 mol of reduced
coenzyme F420(7,8-dimethyl-8-hydroxy-5-deazariboflavin). We report here
on crystal structures of the homotetrameric F420H2 oxidase from Methanothermobacter marburgensis at resolutions of 2.25 Å, 2.25 Å and 1.7 Å,
respectively, from which an active reduced state, an inactive oxidized state
and an active oxidized state could be extracted. As found in structurally
related A-type flavoproteins, the active site is formed at the dimer interface,
where the di-iron center of one monomer is juxtaposed to FMN of the
other. In the active reduced state [Fe(II)Fe(II)FMNH2], the two irons are
surrounded by four histidines, one aspartate, one glutamate and one bridging aspartate. The so-called switch loop is in a closed conformation, thus
preventing F420 binding. In the inactive oxidized state [Fe(III)FMN], the
iron nearest to FMN has moved to two remote binding sites, and the
switch loop is changed to an open conformation. In the active oxidized
state [Fe(III)Fe(III)FMN], both irons are positioned as in the reduced state
but the switch loop is found in the open conformation as in the inactive
oxidized state. It is proposed that the redox-dependent conformational
change of the switch loop ensures alternate complete four-electron O2
reduction and redox center re-reduction. On the basis of the known Si–Si
stereospecific hydride transfer, F420H2 was modeled into the solvent-accessible pocket in front of FMN. The inactive oxidized state might provide the
molecular basis for enzyme inactivation by long-term O2 exposure observed
in some members of the FprA family.
Oxidases catalyze oxidation reactions with O2 as electron acceptor, which is reduced to either H2O2
[E¢(O2 ⁄ H2O2) ¼ + 0.28 V] or H2O [E¢(O2 ⁄ H2O) ¼
+ 0.81 V]. The four-electron reduction of O2 to H2O
generally proceeds without involving O2–, H2O2 or OH
as free intermediates. This is essential, as the superoxide anion radical O2– [E¢(O2– ⁄ H2O2) ¼ + 0.89 V],
H2O2 [E¢(O2 ⁄ H2O2) ¼ + 1.35 V] and the OH radical
[E¢(OH ⁄ H2O) ¼ + 2.3 V] are very strong one-electron oxidants that are highly toxic to living cells, as
shown by the finding that some eukaryotic organisms
deliberately produce these reactive oxygen species via
oxidases to defend themselves against intruding bacteria [1,2].
We have recently discovered in methanogenic Archaea a coenzyme F420H2 oxidase that catalyzes a fourelectron reduction of O2 to H2O, and have provided
evidence that the enzyme is involved in O2 detoxification in these strictly anaerobic microorganisms [3]. In
cell extracts of Methanothermobacter thermoautotrophicus,
Abbreviation
F420, 7,8-dimethyl-8-hydroxy-5-deazariboflavin, coenzyme F420.
1588
FEBS Journal 274 (2007) 1588–1599 ª 2007 The Authors Journal compilation ª 2007 FEBS
F420H2 oxidase is one of the most prominent proteins
[4]. The tetrameric cytoplasmic enzyme is composed of
only one type of subunit, of molecular mass 45 kDa,
and contains, per subunit, one FMN and a di-iron
center. It is specific for coenzyme F420 (7,8-dimethyl-8hydroxy-5-deazariboflavin) as electron donor (apparent
Km ¼ 30 lm) and O2 as electron acceptor (apparent
Km ¼ 2 lm), with an apparent Vmax of the purified
enzyme of 180 s)1 [3]. Coenzyme F420 is a 5-deazaflavin
derivative, and as such transfers hydride anions rather
than single electrons. Upon reduction, 1,5-dihydrocoenzyme F420 is formed, with a prochiral center at C5
(Fig. 1). The F420H2 oxidase has been shown to be Siface stereospecific with respect to C5 of the deazaflavin
[5]. Coenzyme F420 is found in high concentrations only
in methanogenic and sulfate-reducing Archaea.
F420H2 oxidase is not related to other H2O-forming
oxidases such as heme–copper oxidases [6–10], cytochrome bd quinol oxidases [11–14], the multicopper oxidases [15–17], or the apparently only FAD-containing
NADH oxidases from anaerobic bacteria [18–21].
F420H2 oxidase is, however, phylogenetically related to
the A-type flavoprotein family (FprA) [22]. One functionally and structurally characterized member of this
family is the bacterial cytoplasmic NO reductase,
which also contains FMN and a nonheme nonsulfur
di-iron center as prosthetic groups. This enzyme catalyzes the two-electron reduction of 2NO to N2O and
H2O with reduced rubredoxin, but also efficiently catalyzes the four-electron reduction of O2 to 2H2O with
the same one-electron donor [23–28]. Interestingly, the
cytoplasmic NO reductase from Escherichia coli (X-ray
structure unknown) has an extra module at the C-terminus containing a rubredoxin-like center, FMN and
an NADH-binding site [23,29]. In comparison, F420H2
oxidase catalyzes neither the reduction of O2 with
reduced rubredoxin nor the reduction of NO with
F420H2 [3]. This difference in reductant specificity is
surprising for homologous enzymes, considering that
F420 is a deazaflavin (771 Da) that transfers hydride
Fig. 1. Structures of F420H2 and of FMNH2, both viewed from the
Si face. The Re and Si faces of the flavin isoalloxazine ring are
defined relative to C5 of the oxidized deazaflavin F420 [52].
anions at a redox potential (E¢) of ) 360 mV
[30], whereas rubredoxins are iron–sulfur proteins
(6000 Da) that transfer single electrons at redox potentials around 0 ± 100 mV [31].
We report here on the crystal structures of F420H2
oxidase from Methanothermobacter marburgensis in a
reduced state (2.25 Å) and two oxidized states (1.7 Å
and 2.25 Å), and compare them with the 2.5 Å resolution structure of the rubredoxin:NO ⁄ O2 oxidoreductase from Desulfovibrio gigas (31% sequence identity
with F420H2 oxidase) [27] and with the 2.8 Å structure
of the rubredoxin:NO ⁄ O2 oxidoreductase from Moorella thermoacetica (41% sequence identity) [28]. Of
particular interest is the redox state-dependent position
and coordination of the iron atoms and the structural
basis for the specificity of F420H2 oxidase for coenzyme
F420H2 in comparison to that of the two paralogous
enzymes for reduced rubredoxin.
Results and Discussion
Structural basis
F420H2 oxidase from M. marburgensis heterologously
produced in E. coli was isolated and crystallized anaerobically and in the presence of dithiothreitol. Therefore, the isolated enzyme should be in a completely
reduced state with respect to both FMN and the
di-iron center. This assumption is corroborated by the
UV ⁄ visible spectrum of the enzyme, which was typical
for a fully reduced flavoprotein, and by the absence of
an EPR signal, which is consistent with a diferrous or
a diferric center, in which the two irons are antiferromagnetically coupled [24]. The first structure determined at 2.25 Å resolution (Table 1) was based on a
crystal in a monoclinic form (grown in the presence of
F420H2) frozen in liquid nitrogen within the anaerobic
tent. A second and third structure at 2.25 Å and 1.7 Å
resolution (Table 1) were derived from crystals of a
tetragonal and monoclinic crystal form, respectively,
that were frozen in a nitrogen gas stream outside the
anaerobic tent and thus, before freezing, air-exposed
for several minutes at 18 C. We assume that the first
crystal structure reflects an active, predominantly
reduced enzyme state [Fe(II)Fe(II)FMNH2], the second
an inactive oxidized enzyme state [Fe(III)FMN] and
the third an active oxidized [Fe(III)Fe(III)FMN] and
active reduced [Fe(II)Fe(II)FMNH2] state superimposed. Despite considerable efforts, crystals of the
enzyme were not obtained under aerobic conditions.
F420H2 oxidase from M. marburgensis was found in
the crystals ) according to packing considerations ) as a homotetrameric oligomer (Fig. 2A), which
FEBS Journal 274 (2007) 1588–1599 ª 2007 The Authors Journal compilation ª 2007 FEBS
1589
17424658, 2007, 6, Downloaded from https://febs.onlinelibrary.wiley.com/doi/10.1111/j.1742-4658.2007.05706.x by European Molecular Biology Laboratory EMBL, Wiley Online Library on [19/02/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
Structure of di-iron flavoenzyme F420H2 oxidase
H. Seedorf et al.
H. Seedorf et al.
Table 1. Data collection and refinement statistics
Crystallization
Crystal properties
Space group
Cell constants (Å), ()
No. of monomers in
the asymmetric unit
Data collection
Wavelength (Å)
Resolution (Å)
Multiplicity
Completeness (%)
Rsym (%)a
I ⁄ rI (last shell)
Refinement
Rcryst (%)b
Rfree (%)c
No. of reflections
No. of protein atoms
Average B-factor (Å2)
Protein, di-iron, FMN
Bond length rms (Å)
Bond angle rms ()
F420H2 oxidase (anaerobic)
F420H2 oxidase (air-exposed)
F420H2 oxidase (air-exposed)
0.2 M (NH4)2SO4,
0.1 M Mes ⁄ KOH (pH 6.5),
16–22% poly(ethylene glycol)
MME 5000
0.2 M (NH4)2SO4,
0.1 M Mes ⁄ KOH (pH 6.5),
16–22% poly(ethylene glycol)
MME 5000
0.2 M (NH4)2SO4,
0.1 M Mes ⁄ KOH (pH 6.5),
8–16% poly(ethylene glycol)
MME 5000,
15% glycerol
P21
97.8, 123.1, 135.9, 103.4
8
P21
73.7, 120.9, 92.7, 110.4
4
P43212
88.7, 450.4
4
SLS-X10SA
1.000
2.25 (2.3–2.25)
2.6 (2.5)
97.6 (97.8)
6.6 (36.4)
13.7 (1.8)
SLS-X10SA
0.979
1.7 (1.77–1.7)
4.6 (2.1)
99.2 (98.9)
7.8 (41.8)
9.9 (2.3)
SLS-X10SA
0.992
2.25 (2.32–2.25)
4.5 (2.4)
97.2 (74.0)
6.3 (13.7)
16.7 (7.1)
20.6
27.0
103 861
25 349
44.8, 39.4, 27.4
18.6
21.8
156 441
12 652
33.3, 28.9, 20.6
18.8
23.4
80 298
12 652
24.8, 20.6, 15.8
0.011
1.36
0.018
1.87
0.011
1.30
P
P
Rsym¼ |Ii)Ælæ|/ Ii , where Ii is the observed intensity and Ælæ is the averaged intensity obtained from multiple observations of symmetryP
P
related reflections. b Rcryst¼ hkl(|Fobs|)|Fcalc|)/ hkl|Fobs| . c Rcryst where 5% of the observed reflections (randomly selected) are not used for
refinement.
a
is in agreement with previous results (m ¼ 170 kDa)
based on gel filtration experiments [32]. The tetramer
is composed of a loose dimer of two dimers documented by an intradimeric and interdimeric buried surface
of 12% (five ion pairs) and 9.5% (16 ion pairs),
respectively, relative to the entire monomer and dimer
surface areas. Compared to F420H2 oxidase, the interdimer contact areas found in the crystal structures of
rubredoxin:NO ⁄ O2 oxidoreductase from D. gigas
(7.5%; six ion pairs), and of rubredoxin:NO ⁄ O2
oxidoreductase from Mo. thermoacetica (1.2%; no ion
pairs), are smaller, which is in line with their presence
as a dimer in solution [25]. As the catalytically productive oligomeric state is the homodimer (see below),
the differences in quaternary structure may reflect differences in thermoadaptation rather than differences
in function.
The homodimers of the FprA family members reveal
a highly similar architecture, reflected by the rmsd of
about 1.5 Å between the Ca atoms of the monomers,
and by the analogous arrangements of the two mono1590
mers (Fig. 2B). Briefly, each (F420H2 oxidase) monomer is built up of two modules, an N-terminal
b-lactamase-like domain (residues 1–252) harboring a
di-iron center, and a C-terminal flavodoxin-like
domain (residues 253–404) containing FMN. Two
monomers assemble via a head-to-tail arrangement,
such that the b-lactamase and the flavodoxin domains
face each other, thereby forming two separated and
presumably independent active sites (Fig. 2B). Thus, at
the intradimer interface, the di-iron site of one monomer is positioned close to the FMN of the other and
vice versa. Whereas the pyrimidine portion of FMN is
directed to the protein surface, its dimethylbenzyl
group points to the di-iron center. The iron closer to
FMN is, in the following, referred to as proximal iron,
and the other as distal iron. The distance between N5
of FMN and the proximal Fe of about 9 Å is within a
suitable range to allow rapid electron transfer [33]. In
contrast, the di-iron center and the FMN in one
monomer are about 40 Å apart, which is too far for
electron transfer at significant rates (Fig. 2).
FEBS Journal 274 (2007) 1588–1599 ª 2007 The Authors Journal compilation ª 2007 FEBS
17424658, 2007, 6, Downloaded from https://febs.onlinelibrary.wiley.com/doi/10.1111/j.1742-4658.2007.05706.x by European Molecular Biology Laboratory EMBL, Wiley Online Library on [19/02/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
Structure of di-iron flavoenzyme F420H2 oxidase
Fig. 2. Overall structure of F420H2 oxidase. (A) Molecular surface
representation of the tetramer. The tetramer is composed of two
functional dimers, each formed by a head-to-tail arrangement of
two monomers, colored blue ⁄ green and dark gray ⁄ light gray). (B)
Ribbon diagram of the dimer. The monomer is composed of a flavodoxin-like domain (light green ⁄ light blue) harboring FMN (stick
model) and a b-lactamase-like domain (green ⁄ blue), with the di-iron
center depicted as orange spheres. The active sites are located at
the interfaces between two monomers of the functional dimers.
N5 of FMN and the proximal iron (closest to FMN) are sufficiently
close for rapid electron transfer.
Binding of the di-iron center
The di-iron center differs dramatically between the active reduced, inactive oxidized and active oxidized
F420H2 oxidase states (Fig. 3), but also within each
structure, as reflected by differences between the
monomers in the asymmetric unit and by alternative
conformations within one monomer.
In the active reduced enzyme state (present in the
monoclinic crystals frozen in the anaerobic tent and
partly in the air-exposed monoclinic crystals), each
iron ion is tetracoordinated by two imidazole nitrogens
(proximal Fe, His83 and His151; distal Fe, His88 and
His233), one carboxylate (proximal Fe, Glu85; distal
Fe, Asp87), and one bridging carboxylate (Asp170)
(Fig. 3A). Each iron ion contains, approximately trans
to His83 and His88, a fifth coordination site. Both
sites are oriented towards each other and constitute
the dioxygen-binding site (see below). The two Fe(II)
ions are in van der Waals contact with each other,
their distances being 3.5 ± 0.2 Å. The described primary ligation shell essentially corresponds to that
found in the rubredoxin-dependent enzymes. In contrast to the latter enzymes, the average site occupancy
of the proximal iron in F420H2 oxidase is reduced to
approximately 0.4, based on a refinement with equal
temperature factors of the two irons. This finding is in
line with biochemical data that indicate one iron to be
more loosely bound to the enzyme than the other [34].
The low occupancy of the proximal iron leads to an
increase of the temperature factor of its surroundings
but not to a significant alteration of its structure.
In the inactive oxidized state (present in the airexposed tetragonal crystals), the proximal iron is completely absent, and the ligands to iron in the reduced
state have dramatically changed their position, such
that the enzyme is definitively inactive (Fig. 3B). The
side chain of Glu85 is rotated away from the proximal
iron-binding site and constitutes, together with His26
and His267, a new remote metal (iron)-binding site. Its
nature as a metal is compatible with the distance
between the metal and the three ligands of 2.0 Å,
2.1 Å and 2.5 Å, as well as with the height of the electron density peak. Tyr25 evades the new metal-binding
site and becomes hydrogen-bonded to Asp87, which
itself is slightly shifted away from the distal iron. In
other respects, the distal iron-binding site corresponds
to that found in the reduced state. The imidazole
group of His151 ligated to the proximal iron in the
reduced state is shifted by more than 10 Å, and this is
paralleled by a large conformational rearrangement
of the loop between Pro148 and Pro153, referred to in
the following as the switch loop (Fig. 3B). Whereas in
the reduced state this loop is conformationally closed
and directed to the di-iron center and to FMN, in the
oxidized state it flips and creates an open conformation with respect to the accessibility of the redox centers from bulk solvent. Interestingly, the unusual
nonprolyl cis peptide bond formed by Leu150 and
His151 in the reduced state is thereby converted to a
trans peptide bond (Fig. 3B). A nonprolyl cis peptide
bond at this position, which is necessary to project the
imidazolyl ring towards the proximal iron, was also
found in the rubredoxin:NO ⁄ O2 oxidoreductase from
D. gigas but not in the 2.8 Å crystal structure of rubredoxin:NO ⁄ O2 oxidoreductase from Mo. thermoacetica,
possibly due to their low resolution. Unexpectedly, in
the inactive oxidized state, His151, Asp330 and a water
molecule (or a hydroxyl ion) that is hydrogen-bonded to
Arg340 and Lys337 build up another new metal-binding
FEBS Journal 274 (2007) 1588–1599 ª 2007 The Authors Journal compilation ª 2007 FEBS
1591
17424658, 2007, 6, Downloaded from https://febs.onlinelibrary.wiley.com/doi/10.1111/j.1742-4658.2007.05706.x by European Molecular Biology Laboratory EMBL, Wiley Online Library on [19/02/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
Structure of di-iron flavoenzyme F420H2 oxidase
H. Seedorf et al.
H. Seedorf et al.
Fig. 3. Structures of the di-iron-binding site of F420H2 oxidase. The
active site is formed at the homodimer interface, where the di-iron
center of one monomer (green) is juxtaposed to FMN of the other
monomer (blue). Active site amino acid residues and FMN are
shown as stick models, and the two irons as orange spheres.
(A) In the active reduced state, each of the irons is ligated to two
histidines (His83, His88, His151 and His233), one aspartate or glutamate, and one bridging aspartate. The switch loop (red) (a-chain
between Pro148 and Pro153) (the residues are not shown) is in a
closed conformation. Note that His151 projects from the switch
loop towards the proximal iron (closest to FMN), due to a cis peptide bond between Leu150 (not shown) and His151. Trp152 shields
the completely buried di-iron center from bulk solvent (monoclinic
crystal resolved to 2.25 Å). (B) In the inactive oxidized state, the
proximal iron is absent but, alternatively, two new remote metals
are found. The switch loop (black) is in an open conformation. The
proximal iron-ligating residues Glu85, His83 and His151 dramatically
change their conformation; in particular, the last of these moves
more than 10 Å as part of the switch loop (tetragonal crystal
resolved to 2.25 Å). (C) In the active oxidized state, both the proximal and the distal irons are present as in the active reduced state,
but the switch loop adopts an open conformation (black). The active oxidized state is found superimposed with the active reduced
state, such that the closed conformation (red) is also visible in the
electron density map (monoclinic crystal resolved to 1.7 Å).
site located at the protein surface. His83, another
ligand of the proximal iron in the reduced state, is
rotated by about 90 around the Ca–Cb bond, and is
1592
now hydrogen-bonded to the hydroxyl group of
Ser232, which has also changed its conformation
(Fig. 3B). Notably, a conformational change of a histidine ligated to the distal iron was detected in rubredoxin:NO ⁄ O2 oxidoreductase from D. gigas, in
contrast to the rubredoxin:NO ⁄ O2 oxidoreductase
from Mo. thermoacetica [28] and F420H2 oxidase.
A third enzyme state was tentatively extracted from
the electron density of the air-exposed monoclinic crystal, which contains both irons in a similar position and
an occupancy as found in the reduced state. Additionally, Glu85 and Asp87 adopt the conformation of the
reduced state, and the remote metal-binding site is
either not occupied or very little occupied (depending
on the considered monomer in the asymmetric unit).
However, the switch loop reveals electron density not
only for the closed conformation of the reduced state
but also for the open conformation of the inactive
oxidized state, the ratio being 60% to 40%. Consequently, the air-exposed monoclinic crystals includes,
besides the active reduced state, a new superimposed
state referred to as the active oxidized state (Fig. 3C).
The active oxidized state is characterized by a di-iron
center and a switch loop in the open conformation, the
rearrangement from the closed conformation being
presumably triggered by iron oxidation upon air
exposure of the crystals. Therefore, we consider the
active oxidized state as an intermediate of the catalytic
cycle after O2 reduction. Note that the proximal iron
FEBS Journal 274 (2007) 1588–1599 ª 2007 The Authors Journal compilation ª 2007 FEBS
17424658, 2007, 6, Downloaded from https://febs.onlinelibrary.wiley.com/doi/10.1111/j.1742-4658.2007.05706.x by European Molecular Biology Laboratory EMBL, Wiley Online Library on [19/02/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
Structure of di-iron flavoenzyme F420H2 oxidase
is only ligated to Glu85 and Asp170 but not additionally to His83 and His151, as found in the reduced
state.
Redox-dependent changes of the ligation in di-iron
proteins were previously reported for methane monooxygenase reductase hydroxylase [35] and ribonucleotide reductase [36], where, however, only carboxylate
groups of glutamates and aspartates are subject to
conformational alterations.
O2-binding site
The ligand geometry of the di-iron center in the
reduced state offers an attractive O2-binding site within
a pocket coated by the iron-ligating residues Asp87,
Glu85, His151, and His233, as well as by Tyr25,
His26, His175, Phe198 and Leu202 (Fig. 4). In the
eight monomers of the asymmetric unit, the O2-binding pocket is either empty or occupied by a solvent
molecule loosely bound to the distal iron. Whereas in
Fig. 4. The O2-binding site of F420H2 oxidase. Active site amino
acids, FMN and sulfate are shown as stick models. The dioxygenbinding site is surrounded by a pocket coated by residues His233,
Tyr25, His26, His175, Phe198, Asp87, Glu85, His151 and Leu202
(the last four amino acids are not shown). His26 and His175 are
candidates for transferring protons to the peroxo and oxo intermediates (see text). Tyr25 and Phe198 are exchanged in the structurally closely related NO reductases by phenylalanine and tyrosine
(pink). Therefore, Tyr25 and Phe198 are probably responsible for
the finding that F420H2 oxidase does not show NO reductase activity. In the active oxidized state (monomer C), the distal iron is
ligated to a tentatively identified sulfate ion. The two irons are
shown as as orange spheres, and a water molecule as a blue
sphere.
the inactive oxidized state the O2-binding site is destroyed, the electron density map derived from the airexposed monoclinic crystals reveals partial occupation.
In monomers A and B, the extra electron density is
most compatible with a diatomic molecule positioned
slightly closer to the distal than to the proximal iron
and perpendicular to the connection line between the
two irons. In this conformation, one atom ligates to
the proximal and distal irons and the other interacts
with Tyr25 and Asp87. In monomer C, extra electron
density linked to the distal iron is tentatively interpreted as a sulfate ion (Fig. 4). A sulfate anion is
plausible, due to the shape and height of the electron
density peak, the favorable hydrogen bond interactions
with His27 and His175, and the presence of 0.2 m
(NH4)2SO4 in the crystallization buffer. Moreover, an
additional water molecule could be identified between
the two irons and opposite to Asp170. Interestingly,
extra electron density around the distal iron atom suggests an alternative iron position closer to the putative
sulfate ligand due to ligand binding or due to the
altered redox state. Covalent Fe(III)–ligand complexes
are also observed in toluene and methane monooxygenase hydroxylase with acetate, formate and azide as
anion ligands, thereby also corroborating the presence
of the Fe(III) oxidation state [37]. In monomer D, the
water molecule opposite to Asp170 is again visible, but
the electron density connected with the distal iron
could not be reasonably interpreted. The undefined
iron adduct contacts a solvent molecule that is hydrogen-bonded to His26 and His175.
The shape of the O2-binding pocket is approximately
conserved in the structures of rubredoxin:O2 ⁄ NO
oxidoreductases and of F420H2 oxidase, which has no
NO reductase activity. However, the side chains protruding into the pocket partly vary, and might account
for the different specificity. Phe198 in F420H2 oxidase
(Fig. 4) is replaced by tyrosine in the rubredoxindependent enzymes, and the importance of this has
been proven by the decrease of the NO reductase
activity of the Tyr fi Phe mutant in rubredoxin:NO ⁄ O2 reductase [28]. Phe198 in F420H2 oxidase
from M. marburgensis is strictly conserved in other
FprA enzymes from methanogenic Archaea (supplementary Fig. S1), most of which contain at least one
FprA with F420H2 oxidase activity (an exception is
Methanopyrus kandleri). Another crucial residue is
Tyr25 (Fig. 4), which is invariant in methanogenic
Archaea and replaced by a phenylalanine in the rubredoxin-dependent enzymes. It protrudes from a loop
variable within the FprA family, and its hydroxyl
group interacts with the Fe-ligating carboxylate group
of Glu85 and Asp87. The side chain of Tyr25 is in van
FEBS Journal 274 (2007) 1588–1599 ª 2007 The Authors Journal compilation ª 2007 FEBS
1593
17424658, 2007, 6, Downloaded from https://febs.onlinelibrary.wiley.com/doi/10.1111/j.1742-4658.2007.05706.x by European Molecular Biology Laboratory EMBL, Wiley Online Library on [19/02/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
Structure of di-iron flavoenzyme F420H2 oxidase
H. Seedorf et al.
H. Seedorf et al.
der Waals contact with the putative ligand in the
O2-binding site, and it might be speculated that its
hydroxyl group interferes with the bulky N2O, thus
preventing its formation.
Binding of FMN and modeling of F420H2
The conformation and binding characteristics of FMN
are nearly identical in all of the analyzed structures of
F420H2 oxidase but also in comparison to those of
other members of the FprA family. However, the specific FMN–polypeptide interactions can be most accurately described in F420H2 oxidase, due to the higher
resolution. FMN has an essentially planar isoalloxazine ring (Fig. 5), which is compatible with FMN
being in either the reduced or the oxidized state [38]. A
large number of polar contacts are formed between the
peptide nitrogens of Met266, His267, Gly268, Ser269,
Thr270, Tyr319, Asp320, Gly353, and Gly354, as well
as Gly356 and the pyrimidine and phosphate components of FMN, indicating a rigid binding mode.
Whereas the Re face of the ring is attached to residues
Thr317, Ile318, Tyr319 and Met266 of the flavodoxinlike domain, the Si face is solvent-accessible, and a
water-filled pocket is placed between the isalloxazine
ring and the opposite monomer (Fig. 5). This pocket
can be reliably considered as the F420H2-binding site,
although the experimental verification by structure
determination of an enzyme–F420 complex was not
Fig. 5. The F420H2-binding site of F420H2 oxidase in the active oxidized state. F420H2 (yellow stick model) is modeled into its binding
pocket with its Si face oriented towards the Si face of FMN (blue
stick model). C5 of F420H2 and N5 of FMN, between which the
hydride is transferred, are positioned within the van der Waals distance (approximately 3 Å). In this conformation, the Re face of
F420H2 is attached to the switch loop in the open conformation
(black), and the pyrimidine group of F420 reaches the di-iron center.
1594
feasible. Remarkably, solely in the oxidized state, the
available space in front of the Si face of the FMN ring
is sufficient to accommodate the bulky deazaisoalloxazine ring of F420H2 (open conformation), whereas in
the reduced state (closed conformation) the switch
loop is directed towards the prosthetic groups, and the
bulky side chains of His151 and Trp152 block F420H2
binding.
Model building of F420H2 was governed by the
experimentally determined Si-face stereospecificity of
the hydride transfer to and from F420 [5], which defines
the orientation of the deazaflavin relative to the FMN
face, by the assumed aromatic stacking interactions
between the two ring systems observed in various
systems [39,40], and by the required proximity between
C5 of F420H2 and N5 of FMN (Fig. 5), implying that
the generated complex is competent for hydride transfer [40]. Thus positioned, the tricyclic F420 ring is sandwiched between the isoalloxazine ring of FMN and the
segment between His151 and Pro153 of the switch
loop, whereby the imidazole group of His151 interacts
with the bottom of F420H2 and the side chain of
Trp152 with its face (Fig. 5). The crucial residue
Trp152 is kept in place by a hydrogen bond between
its indole nitrogen atom and the hydroxyl group of
Tyr319. The l-lactyl-l-glutamyl-l-glutamic acid phosphodiester portion of F420 (see Fig. 1) was placed at
the interface between the subunits such that its phosphate group is anchored by His117 and His267, which
are both strictly conserved, and its first carboxylate
group by Lys272. In this conformation, the mentioned
F420H2 portion replaces a water chain that extends
from the Si side of FMN to the bulk solvent, and
therefore requires only minor displacements of the
polypeptide (Fig. 5).
In the crystal structures of rubredoxin:NO ⁄ O2
oxidoreductases from D. gigas and of rubredoxin:
NO ⁄ O2 oxidoreductase from Mo. thermoacetica, the
pocket is filled up from the entrance side by the side
chains of Trp347 and Met146, which are both conserved in the rubredoxin-dependent enzymes but
replaced by an asparagine and a leucine in F420H2
oxidase (supplementary Figs S1 and S2). F420H2 cannot enter the pocket, and this effectively precludes
direct interaction of this electron donor with the FMN
of the active site. On the other hand, where and how
rubredoxin with a molecular mass of approximately
6 kDa binds to the two rubredoxin-dependent enzymes
and not to F420H2 oxidase is not yet known. The mentioned Trp347 would be a candidate for shuttling electrons from rubredoxin to FMN.
The structure-based analysis of the substrate binding
in F420H2 oxidase teaches us once again that, on the
FEBS Journal 274 (2007) 1588–1599 ª 2007 The Authors Journal compilation ª 2007 FEBS
17424658, 2007, 6, Downloaded from https://febs.onlinelibrary.wiley.com/doi/10.1111/j.1742-4658.2007.05706.x by European Molecular Biology Laboratory EMBL, Wiley Online Library on [19/02/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
Structure of di-iron flavoenzyme F420H2 oxidase
Structure of di-iron flavoenzyme F420H2 oxidase
basis of sequence homology, the function of proteins
cannot be inferred even if their crystal structures are
known in detail. In the FprA family, the electron
donor and acceptor specificity and the accompanied
redox mechanisms are totally different, although the
structural framework, the binding mode of FMN and
the di-iron center, as well as the electron transfer process, are strictly conserved. As discussed in detail, only
a few side chain exchanges are sufficient to prevent or
allow NO versus O2 as electron donor and to block or
favor F420H2 binding over FMN.
The catalytic reaction
The F420H2 oxidase reaction represents a ping-pong
process where, in a first reaction, four electrons from
the diferrous di-iron and FMNH2 are transferred to
the dioxygen, thereby forming two water molecules
without the release of reactive oxygen species, and in a
second reaction, the two redox centers are re-reduced
by two hydride transfer reactions between F420H2 and
FMN. The first half-cycle is assumed to begin with the
FMN and the di-iron center of the enzyme both in
the fully reduced state [Fe(II)Fe(II)FMNH2], for which
the structure has been established. As a first step, the
enzyme binds one molecule of O2 transiently, forming
a peroxo intermediate bridging the two iron atoms, as
suggested by mechanistic studies with di-iron(II) complexes [41,42]. Then, a first water molecule is released,
leaving behind the enzyme in the diferric l-O(H)
FMNH2 state (reaction in Scheme 1).
Fe(II)Fe(II)FMNH2 þ O2 þ 2 Hþ
! Fe(III)OFe(III)FMNH2 þ H2 O
ðScheme 1Þ
Then, two electrons are transferred from the reduced
FMN to the l-O(H) bridge between the two irons in
the diferric state, with the release of the second water
molecule (reaction in Scheme 2).
Fe(III)OFe(III)FMNH2 ! Fe(III)Fe(III)FMN þ H2 O
ðScheme 2Þ
We assume that the generated Fe(III)Fe(III)FMN state
is reflected in the active oxidized structure. The second
half-cycle proceeds with binding of the first F420H2
and subsequent reduction of FMN, from which the
electrons are shuttled one by one to the irons. After
release of F420, a second F420H2 binds, reduces FMN
and leaves the active site (reactions in Schemes 3
and 4).
Fe(III)Fe(III)FMN þ F420 H2
! Fe(II)Fe(II)FMN þ F420 þ 2 Hþ
ðScheme 3Þ
Fe(II)Fe(II)FMN þ F420 H2 ! Fe(II)Fe(II)FMNH2 þ F420
ðScheme 4Þ
The enzyme is now back in the reduced FMN and
diferrous state. Electron transfer between the reduced
FMN and the proximal iron across the homodimeric
subunit interface is most likely mediated via the dimethylbenzyl group of FMN and His151 or Asp85
(Fig. 3A). Both residues have a minimal distance to
C8 of the flavin ring of 3.7 Å. Trp152 and Tyr319,
flanking the mentioned residues, might additionally
support a rapid electron transfer process between the
reactions in Schemes 1 and 2. Proton transfer to the
peroxo and oxo intermediates generated during oxygen
reduction might be directly or indirectly accomplished
by the strictly conserved residues His26 and His175,
which are both accessible to bulk solvent (Fig. 4). In
the reduced and active oxidized state, the two pronounced histidines are too far away (4.0–4.5 Å) from
the O2-binding site, and a water molecule visible in the
electron density map between their side chains (in
monomer D) might be used as mediator. However,
His26 can be positioned in hydrogen bond contact
with a tentatively modeled O2 upon minor structural
rearrangements, as seen in the inactive oxidized state
(Fig. 3B).
Experimental evidence is provided that the FprA
oxidase reaction avoids the release of reactive oxygen
species [29], which requires a direct and controlled
four-electron reduction of O2. Structural data suggest
that the sequential course of the complete O2 reduction and the complete prosthetic group re-reduction
are ensured by the redox-dependent conformation of
the switch loop (Fig. 3C). In the case that the di-iron
center and FMN are reduced, the side chains of the
key residues His151 and Trp152, protruding from the
switch loop, complete the di-iron center for O2 activation and block the access of F420H2 (which is compatible with the unsuccessful cocrystallization experiments
with F420H2 oxidase in the reduced state and F420H2).
When the prosthetic group becomes oxidized upon O2
reduction, the switch loop is rearranged, thereby abolishing the catalytic competence of the di-iron center
and allowing the binding of F420H2 and the subsequent hydride transfer. A hypothetical scenario might
be that iron oxidation weakens the interactions
between the proximal iron and His151, leading to an
energetically favorable cis–trans isomerization of the
peptide bond between Leu150 and His151, thereby
inducing the structural rearrangement of the switch
loop. For comparison, a stepwise O2 reduction is realized in a related iron–sulfur and flavin-containing ferredoxin oxidase found in methanogenic Archaea, but
FEBS Journal 274 (2007) 1588–1599 ª 2007 The Authors Journal compilation ª 2007 FEBS
1595
17424658, 2007, 6, Downloaded from https://febs.onlinelibrary.wiley.com/doi/10.1111/j.1742-4658.2007.05706.x by European Molecular Biology Laboratory EMBL, Wiley Online Library on [19/02/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
H. Seedorf et al.
H. Seedorf et al.
also in other anaerobic prokaryotes, that catalyze the
reduction of O2 to H2O with H2O2 as free intermediate [43]. Interestingly, despite its completely different
O2 activation mechanism, several architectural features
are common to those described for the FprA family,
such as its homotetrameric organization, its head-totail arrangement of two monomers juxtaposing FMN
and the [4Fe)4S] cluster from two different monomers, and the similar fold of the FMN-binding
domain [44].
The outlined mechanism provides no functional role
for the inactive oxidized state structurally characterized
for F420H2 oxidase. However, it is conceivable that the
displacement of the proximal iron to the remote metalbinding sites over a distance of about 6 Å and 15 Å
(Fig. 3B) is related to the inactivation of rubredoxindependent NO reductases after multiple O2 reduction
cycles. A shift of the proximal iron would be energetically plausible, as its fixation by ligands is reduced in
the active oxidized state, and because it can move concomitantly with the swinging side chain of Glu85 to
constitute, with His26 and His267, an efficient metalbinding site. Inactivation of FprAs in the presence of
large amounts of O2 might be biologically useful, as
the cell would lose reducing power without eventually
getting rid of the oxygen.
Experimental procedures
Purification and crystallization
The fprA gene from M. marburgensis (DSMZ2133) was
overexpressed in E. coli as described, except that the cells
were grown in 2 L of trypton ⁄ phosphate medium rather
than in LB medium [3,5]. Purification was performed under
exclusion of oxygen in an anaerobic chamber (Coy) filled
with 95% N2 ⁄ 5% H2 (v ⁄ v) and containing a palladium catalyst for O2 reduction with H2. Initial trials to crystallize
F420H2 oxidase were performed with the hanging ⁄ sittingdrop, vapor-diffusion method using Basic and Extension
crystallization kits from Sigma-Aldrich (Sigma-Aldrich,
St Louis, USA). For the screens, 2 lL of the enzyme solution (containing 20 mgÆmL)1 of F420H2 oxidase) and 2 lL
of reservoir solutions were mixed and incubated at 4 C.
Under aerobic conditions, crystals of FprA were not
observed. However, under anaerobic conditions and in the
presence of 1 mm dithiothreitol, crystals were obtained at
10 C using 0.2 m (NH4)2SO4, 0.1 m Mes ⁄ KOH (pH 6.5)
and 30% poly(ethylene glycol) [30% poly(ethylene glycol)
monomethylether 5000] (MME) 5000 or 0.2 m Mgformate. Optimization of crystal quality, mainly varying the
drop size (20 lL), precipitant concentrations and additional
agents, resulted in three different crystal forms (see Table 1).
1596
Data collection, structure determination and
refinement
Data were collected at the beam line X10SA of the SwissLight-Source (Villigen, Switzerland) from anaerobically
grown crystals, the first kept in an oxygen-free atmosphere
and the second exposed to air. Processing and scaling were
performed with the hkl [45] and xds [46] packages. The
quality of the data and crystallographic parameters are
summarized in Table 1. The structure of the enzyme based
on the air-exposed monoclinic crystals was solved by
molecular replacement using epmr [47] based on the 2.8 Å
structure of rubredoxin:NO ⁄ O2 oxidoreductase from
Mo. thermoacetica [28]. Using the 2.5 Å structure of rubredoxin:NO ⁄ O2 oxidoreductase from D. gigas [27] gave less
reliable results, although the structures of the two rubredoxin-dependent enzymes are very similar, with respect to
both the primary structure (42% sequence identity) and the
quaternary structure (rmsd 1.3 Å for the Ca atoms of the
two models). The phases for the other two crystals were
obtained by molecular replacement using the model from
the air-exposed monoclinic crystals [47]. Refinement of the
structures based on crystals were performed using o [48]
and cns [49], applying the four-fold noncrystallographic
symmetry (NCS) relationship for the lower resolution data.
Refinement was completed with the program refmac5 [50],
using the TLS option (each monomer was treated as a separate TLS group), maximum likelihood minimization and
isotropic B-value refinement. The refinement statistics are
given in Table 1. Except for the C-terminal arginine, the
entire polypeptide chain is visible in the electron density
map. The stereochemical quality of the model was checked
with the program procheck [51]. Figures 2–5 were generated with pymol (http://www.pymol.org). The coordinates
of the structures based on anaerobically treated crystals, on
air-exposed monoclinic crystals and tetragonal crystals are
deposited in the Protein Data Bank (http://www.rcsb.org)
with accession numbers 2OHI, 2OHH and 2OHJ, respectively.
Acknowledgements
This work was supported by the Max Planck Society
and by the Fonds der Chemischen Industrie. We thank
Hartmut Michel for continuous support, and the staff
of the X10SA beamline at the Swiss-Light-Source,
Villigen for assistance during data collection.
References
1 Fang FC (2004) Antimicrobial reactive oxygen and
nitrogen species: concepts and controversies. Nat Rev
Microbiol 2, 820–832.
FEBS Journal 274 (2007) 1588–1599 ª 2007 The Authors Journal compilation ª 2007 FEBS
17424658, 2007, 6, Downloaded from https://febs.onlinelibrary.wiley.com/doi/10.1111/j.1742-4658.2007.05706.x by European Molecular Biology Laboratory EMBL, Wiley Online Library on [19/02/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
Structure of di-iron flavoenzyme F420H2 oxidase
2 El-Benna J, Dang PMC, Gougerot-Pocidalo MA &
Elbim C (2005) Phagocyte NADPH oxidase: a multicomponent enzyme essential for host defenses. Arch
Immunol Ther Exp 53, 199–206.
3 Seedorf H, Dreisbach A, Hedderich R, Shima S &
Thauer RK (2004) F420H2 oxidase (FprA) from Methanobrevibacter arboriphilus, a coenzyme F420-dependent
enzyme involved in O2 detoxification. Arch Microbiol
182, 126–137.
4 Farhoud MH, Wessels HJ, Steenbakkers PJ, Mattijssen
S, Wevers RA, van Engelen BG, Jetten MS, Smeitink
JA, van den Heuvel LP & Keltjens JT (2005) Protein
complexes in the archaeon Methanothermobacter thermautotrophicus analyzed by blue native ⁄ SDS-PAGE and
mass spectrometry. Mol Cell Proteomics 4, 1653–1663.
5 Seedorf H, Kahnt J, Pierik AJ & Thauer RK (2005)
Si-face stereospecificity at C5 of coenzyme F420 for
F420H2 oxidase from methanogenic Archaea as determined by mass spectrometry. FEBS J 272, 5337–5342.
6 Michel H, Behr J, Harrenga A & Kannt A (1998) Cytochrome C oxidase: structure and spectroscopy. Annu
Rev Biophys Biomed 27, 329–356.
7 Giuffre A, Stubauer G, Sarti P, Brunori M, Zumft WG,
Buse G & Soulimane T (1999) The heme-copper oxidases of Thermus thermophilus catalyze the reduction of
nitric oxide: evolutionary implications. Proc Natl Acad
Sci USA 96, 14718–14723.
8 Brunori M, Giuffre A & Sarti P (2005) Cytochrome c
oxidase, ligands and electrons. J Inorg Biochem 99, 324–
336.
9 Zumft WG (2005) Nitric oxide reductases of prokaryotes with emphasis on the respiratory, heme-copper oxidase type. J Inorg Biochem 99, 194–215.
10 Branden G, Pawate AS, Gennis RB & Brzezinski P
(2006) Controlled uncoupling and recoupling of proton
pumping in cytochrome c oxidase. Proc Natl Acad Sci
USA 103, 317–322.
11 Hill BC, Hill JJ & Gennis RB (1994) The room-temperature reaction of carbon-monoxide and oxygen with
the cytochrome bd quinol oxidase from Escherichia coli.
Biochemistry 33, 15110–15115.
12 Dmello R, Hill S & Poole RK (1996) The cytochrome
bd quinol oxidase in Escherichia coli has an extremely
high oxygen affinity and two oxygen-binding haems:
implications for regulation of activity in vivo by oxygen
inhibition. Microbiol UK 142, 755–763.
13 Zhang J, Hellwig P, Osborne JP, Huang HW, MoenneLoccoz P, Konstantinov AA & Gennis RB (2001) Sitedirected mutation of the highly conserved region near
the Q-loop of the cytochrome bd quinol oxidase from
Escherichia coli specifically perturbs heme b(595). Biochemistry 40, 8548–8556.
14 Zhang J, Barquera B & Gennis RB (2004) Gene fusions
with beta-lactamase show that subunit I of the cytochrome bd quinol oxidase from E. coli has nine
Structure of di-iron flavoenzyme F420H2 oxidase
15
16
17
18
19
20
21
22
23
24
25
26
transmembrane helices with the O2 reactive site near the
periplasmic surface. FEBS Lett 561, 58–62.
Lee SK, George SD, Antholine WE, Hedman B, Hodgson KO & Solomon EI (2002) Nature of the intermediate formed in the reduction of O2 to H2O at the
trinuclear copper active site in native laccase. J Am
Chem Soc 124, 6180–6193.
Johnson DL, Thompson JL, Brinkmann SM, Schuller
KA & Martin LL (2003) Electrochemical characterization of purified Rhus vernicifera laccase: voltammetric
evidence for a sequential four-electron transfer. Biochemistry 42, 10229–10237.
Riva S (2006) Laccases: blue enzymes for green chemistry. Trends Biotechnol 24, 219–226.
Ross RP & Claiborne A (1992) Molecular-cloning and
analysis of the gene encoding the NADH oxidase from
Streptococcus faecalis 10c1 ) comparison with NADH
peroxidase and the flavoprotein disulfide reductases.
J Mol Biol 227, 658–671.
Matsumoto J, Higuchi M, Shimada M, Yamamoto Y &
Kamio Y (1996) Molecular cloning and sequence analysis of the gene encoding the H2O-forming NADH oxidase from Streptococcus mutans. Biosci Biotechnol
Biochem 60, 39–43.
Brown DM, Upcroft JA & Upcroft P (1996) A H2Oproducing NADH oxidase from the protozoan parasite
Giardia duodenalis. Eur J Biochem 241, 155–161.
Kawasaki S, Ishikura J, Chiba D, Nishino T & Niimura
Y (2004) Purification and characterization of an H2Oforming NADH oxidase from Clostridium aminovalericum: existence of an oxygen-detoxifying enzyme in an
obligate anaerobic bacteria. Arch Microbiol 181, 324–
330.
Wasserfallen A, Ragettli S, Jouanneau Y & Leisinger T
(1998) A family of flavoproteins in the domains Archaea
and Bacteria. Eur J Biochem 254, 325–332.
Gomes CM, Giuffre A, Forte E, Vicente JB, Saraiva
LM, Brunori M & Teixeira M (2002) A novel type of
nitric-oxide reductase. Escherichia coli flavorubredoxin.
J Biol Chem 277, 25273–25276.
Silaghi-Dumitrescu R, Coulter ED, Das A, Ljungdahl
LG, Jameson GNL, Huynh BH & Kurtz DM (2003)
A flavodiiron protein and high molecular weight
rubredoxin from Moorella thermoacetica with
nitric oxide reductase activity. Biochemistry 42, 2806–
2815.
Silaghi-Dumitrescu R, Kurtz DM, Ljungdahl LG &
Lanzilotta WN (2005a) X-ray crystal structures of
Moorella thermoacetica FprA. Novel diiron site structure and mechanistic insights into a scavenging nitric
oxide reductase. Biochemistry 44, 6492–6501.
Rodrigues R, Vicente JB, Felix R, Oliveira S, Teixeira
M & Rodrigues-Pousada C (2006) Desulfovibrio gigas
flavodiiron protein affords protection against nitrosative
stress in vivo. J Bacteriol 188, 2745–2751.
FEBS Journal 274 (2007) 1588–1599 ª 2007 The Authors Journal compilation ª 2007 FEBS
1597
17424658, 2007, 6, Downloaded from https://febs.onlinelibrary.wiley.com/doi/10.1111/j.1742-4658.2007.05706.x by European Molecular Biology Laboratory EMBL, Wiley Online Library on [19/02/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
H. Seedorf et al.
H. Seedorf et al.
27 Frazao C, Silva G, Gomes CM, Matias P, Coelho R,
Sieker L, Macedo S, Liu MY, Oliveira S, Teixeira M
et al. (2000) Structure of a dioxygen reduction enzyme
from Desulfovibrio gigas. Nat Struct Biol 7, 1041–1045.
28 Silaghi-Dumitrescu R, Ng KY, Viswanathan R & Kurtz
DM (2005b) A flavo-diiron protein from Desulfovibrio
vulgaris with oxidase and nitric oxide reductase activities. Evidence for an in vivo nitric oxide scavenging
function. Biochemistry 44, 3572–3579.
29 Gomes CM, Vicente JB, Wasserfallen A & Teixeira M
(2000) Spectroscopic studies and characterization of a
novel electron-transfer chain from Escherichia coli involving a flavorubredoxin and its flavoprotein reductase
partner. Biochemistry 39, 16230–16237.
30 Thauer RK (1998) Biochemistry of methanogenesis: a
tribute to Marjory Stephenson. Microbiology 144, 2377–
2406.
31 Lin IJ, Gebel EB, Machonkin TE, Westler WM &
Markley JL (2005) Changes in hydrogen-bond strengths
explain reduction potentials in 10 rubredoxin variants.
Proc Natl Acad Sci USA 102, 14581–14586.
32 Wasserfallen A, Huber K & Leisinger T (1995) Purification and structural characterization of a flavoprotein
induced by iron limitation in Methanobacterium thermoautotrophicum Marburg. J Bacteriol 177, 2436–2441.
33 Page CC, Moser CC, Chen XX & Dutton PL (1999)
Natural engineering principles of electron tunnelling in
biological oxidation–reduction. Nature 402, 47–52.
34 Seedorf H (2003) F420H2-Oxidase, ein neuartiges vier
Elektronen umsetzendes Flavoprotein in methanogenen
Archaea. Diploma Thesis, Philipps-University,
Marburg.
35 Rudd DJ, Sazinsky MH, Merkx M, Lippard SJ, Hedman B & Hodgson KO (2004) Determination by X-ray
absorption spectroscopy of the Fe–Fe separation in the
oxidized form of the hydroxylase of methane monooxygenase alone and in the presence of MMOD. Inorg
Chem 43, 4579–4589.
36 Sintchak MD, Arjara G, Kellogg BA, Stubbe J &
Drennan CL (2002) The crystal structure of class II
ribonucleotide reductase reveals how an allosterically
regulated monomer mimics a dimer. Nat Struct Biol
9, 293–300.
37 Sazinsky MH & Lippard SJ (2006) Correlating structure
with function in bacterial multicomponent monooxygenases and related diiron proteins. Acc Chem Res 39,
558–566.
38 Moonen CTW, Vervoort J & Muller F (1984) Reinvestigation of the structure of oxidized and reduced flavin:
carbon-13 and nitrogen-15 nuclear magnetic resonance
study. Biochemistry 23, 4859–4867.
39 Warkentin E, Mamat B, Sordel-Klippert M, Wicke M,
Thauer RK, Iwata M, Iwata S, Ermler U & Shima S
(2001) Structures of F420H2:NADP+ oxidoreductase
1598
40
41
42
43
44
45
46
47
48
49
50
51
52
with and without its substrates bound. EMBO J 20,
6561–6569.
Pejchal R, Sargeant R & Ludwig ML (2005) Structures
of NADH and CH3-H4 folate complexes of Escherichia
coli methylenetetrahydrofolate reductase reveal a Spartan strategy for a ping-pong reaction. Biochemistry 44,
11447–11457.
Kryatov SV, Rybak-Akimova EV, MacMurdo VL &
Que L Jr (2001) A mechanistic study of the reaction
between a diiron(II) complex [FeII(2)(mu-OH)2(6-Me3TPA)2](2+) and O2 to form a diiron(III) peroxo complex. Inorg Chem 40, 2220–2228.
Costas M, Cady CW, Kryatov SV, Ray M, Ryan MJ,
Rybak-Akimova EV & Que L Jr (2003) Role of carboxylate bridges in modulating nonheme diiron(II) ⁄ O(2)
reactivity. Inorg Chem 42, 7519–7530.
Cruz F & Ferry JG (2006) Interaction of iron–sulfur
flavoprotein with oxygen and hydrogen peroxide. BBA
Gen Subjects 1760, 858–864.
Andrade SL, Cruz F, Drennan CL, Ramakrishnan V,
Rees DC, Ferry JG & Einsle O (2005) Structures of the
iron–sulfur flavoproteins from Methanosarcina thermophila and Archaeoglobus fulgidus. J Bacteriol 187, 3848–
3854.
Otwinowski Z & Minor W (1997) Processing of X-ray
diffraction data collected in oscillation mode. Methods
Enzymol 276, 307–326.
Kabsch W (1993) Automatic processing of rotation
diffraction data from crystals of initially unknown
symmetry and cell constants. J Appl Crystallogr 26,
795–800.
Kissinger CR, Gehlhaar DK & Fogel DB (1999) Rapid
automated molecular replacement by evolutionary
search. Acta Crystallogr D 55, 484–491.
Jones TA, Zou JY, Cowan SW & Kjeldgaard M (1991)
Improved methods for building protein models in electron density maps and the location of errors in these
models. Acta Crystallogr A 47(2), 110–119.
Brünger AT, Adams PD, Clore GM, DeLano WL, Gros
P, Grosse-Kunstleve RW, Jiang JS, Kuszewski J, Nilges
M, Pannu NS et al. (1998) Crystallography & NMR
system: a new software suite for macromolecular
structure determination. Acta Crystallogr D 54,
905–921.
Murshudov GN, Vagin AA & Dodson EJ (1997)
Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallogr D 53, 240–
255.
Laskowski RA, MacArthur MW, Moss DS & Thornton
JM (1993) PROCHECK: a program to check the stereochemical quality of protein structures. J Appl Crystallogr 26, 283–291.
Sumner JS & Matthews RG (1992) Stereochemistry and
mechanism of hydrogen transfer between NADPH and
FEBS Journal 274 (2007) 1588–1599 ª 2007 The Authors Journal compilation ª 2007 FEBS
17424658, 2007, 6, Downloaded from https://febs.onlinelibrary.wiley.com/doi/10.1111/j.1742-4658.2007.05706.x by European Molecular Biology Laboratory EMBL, Wiley Online Library on [19/02/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
Structure of di-iron flavoenzyme F420H2 oxidase
methylenetetrahydrofolate in the reaction catalyzed by
methylenetetrahydrofolate reductase from pig liver.
J Am Chem Soc 114, 6949–6959.
Supplementary material
The following supplementary material is available
online:
Fig. S1. Sequence alignment of F420H2 oxidase from
Methanothermobacter marburgensis, rubredoxin:NO ⁄ O2
oxidoreductase from Desulfovibrio gigas, rubredoxin:
NO ⁄ O2 oxidoreductase from Moorella thermoacetica,
F420H2 oxidase from Methanobrevibacter arboriphilus,
and other FprAs from methanogenic archaea assumed
to have F420H2 oxidase activity. The amino acids
involved in FMN binding are highlighted in yellow,
and those involved in iron coordination are highlighted
in red. The amino acids lining the cavity above the
di-iron center are given in blue. The prominent trypto-
Structure of di-iron flavoenzyme F420H2 oxidase
phan between FMN and the di-iron site is in green.
The two amino acids linked via a cis peptide bond are
indicated by asterisks. Other amino acids conserved in
all sequences are highlighted in gray.
Fig. S2. Structures of the F420H2 pocket at the interface of two subunits in the functional dimer of (A)
F420H2 oxidase from Methanothermobacter marburgensis, (B) rubredoxin:NO ⁄ O2 oxidoreductase from
Desulfovibrio gigas, and (C) rubredoxin:NO ⁄ O2 oxidoreductase from Moorella thermoacetica.
This material is available as part of the online article
from http://www.blackwell-synergy.com
Please note: Blackwell Publishing is not responsible for
the content or functionality of any supplementary
materials supplied by the authors. Any queries (other
than missing material) should be directed to the corresponding author for the article.
FEBS Journal 274 (2007) 1588–1599 ª 2007 The Authors Journal compilation ª 2007 FEBS
1599
17424658, 2007, 6, Downloaded from https://febs.onlinelibrary.wiley.com/doi/10.1111/j.1742-4658.2007.05706.x by European Molecular Biology Laboratory EMBL, Wiley Online Library on [19/02/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
H. Seedorf et al.