Báo cáo khoa học: Structure of coenzyme F420H2 oxidase (FprA), a di-iron flavoprotein from methanogenic Archaea catalyzing the reduction of O2 to H2O ppt

Structure of coenzyme F
420
H
2
oxidase (FprA), a di-iron
flavoprotein from methanogenic Archaea catalyzing the
reduction of O
2
to H
2
O
Henning Seedorf
1
, Christoph H. Hagemeier
1
, Seigo Shima
1
, Rudolf K. Thauer
1
,
Eberhard Warkentin
2
and Ulrich Ermler
2
1 Max Planck Institute for Terrestrial Microbiology, Marburg, Germany
2 Max Planck Institute for Biophysics, Frankfurt am Main, Germany
Oxidases catalyze oxidation reactions with O
2
as elec-
tron acceptor, which is reduced to either H
2

O
2
[E°¢(O
2
⁄ H
2
O
2
) ¼ + 0.28 V] or H
2
O[E°¢(O
2
⁄ H
2
O) ¼
+ 0.81 V]. The four-electron reduction of O
2
to H
2
O
generally proceeds without involving O
2
–
,H
2
O
2
or OH
as free intermediates. This is essential, as the superox-
ide anion radical O

2
–
[E°¢(O
2
–
⁄ H
2
O
2
) ¼ + 0.89 V],
H
2
O
2
[E°¢(O
2
⁄ H
2
O
2
) ¼ + 1.35 V] and the OH radical
[E°¢(OH ⁄ H
2
O) ¼ + 2.3 V] are very strong one-elec-
tron 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 bac-
teria [1,2].
We have recently discovered in methanogenic Arch-

aea a coenzyme F
420
H
2
oxidase that catalyzes a four-
electron reduction of O
2
to H
2
O, and have provided
evidence that the enzyme is involved in O
2
detoxifica-
tion in these strictly anaerobic microorganisms [3]. In
cell e xtracts of Methanothermobacter t hermoautotrophicus,
Keywords
coenzyme F
420
; crystal structure; di-iron
center; F
420
H
2
oxidase; O
2
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:
(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 F
420
H
2
oxidase found in methanogenic Archaea
catalyzes the four-electron reduction of O
2
to 2H
2
O with 2 mol of reduced
coenzyme F
420
(7,8-dimethyl-8-hydroxy-5-deazariboflavin). We report here
on crystal structures of the homotetrameric F
420
H
2
oxidase from Methan-
othermobacter marburgensis at resolutions of 2.25 A
˚
, 2.25 A
˚
and 1.7 A
˚

,
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)FMNH
2
], the two irons are
surrounded by four histidines, one aspartate, one glutamate and one brid-
ging aspartate. The so-called switch loop is in a closed conformation, thus
preventing F
420
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 O
2
reduction and redox center re-reduction. On the basis of the known Si–Si
stereospecific hydride transfer, F
420
H
2
was modeled into the solvent-access-
ible pocket in front of FMN. The inactive oxidized state might provide the
molecular basis for enzyme inactivation by long-term O
2
exposure observed

in some members of the FprA family.
Abbreviation
F
420
, 7,8-dimethyl-8-hydroxy-5-deazariboflavin, coenzyme F
420
.
1588 FEBS Journal 274 (2007) 1588–1599 ª 2007 The Authors Journal compilation ª 2007 FEBS
F
420
H
2
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 F
420
(7,8-dimethyl-8-
hydroxy-5-deazariboflavin) as electron donor (apparent
K
m
¼ 30 lm) and O
2
as electron acceptor (apparent
K
m
¼ 2 lm ), with an apparent V
max
of the purified

enzyme of 180 s
)1
[3]. Coenzyme F
420
is a 5-deazaflavin
derivative, and as such transfers hydride anions rather
than single electrons. Upon reduction, 1,5-dihydroco-
enzyme F
420
is formed, with a prochiral center at C5
(Fig. 1). The F
420
H
2
oxidase has been shown to be Si-
face stereospecific with respect to C5 of the deazaflavin
[5]. Coenzyme F
420
is found in high concentrations only
in methanogenic and sulfate-reducing Archaea.
F
420
H
2
oxidase is not related to other H
2
O-forming
oxidases such as heme–copper oxidases [6–10], cyto-
chrome bd quinol oxidases [11–14], the multicopper ox-
idases [15–17], or the apparently only FAD-containing

NADH oxidases from anaerobic bacteria [18–21].
F
420
H
2
oxidase is, however, phylogenetically related to
the A-type flavoprotein family (FprA) [22]. One func-
tionally 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 cata-
lyzes the two-electron reduction of 2NO to N
2
O and
H
2
O with reduced rubredoxin, but also efficiently cata-
lyzes the four-electron reduction of O
2
to 2H
2
O 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-ter-
minus containing a rubredoxin-like center, FMN and
an NADH-binding site [23,29]. In comparison, F
420
H
2

oxidase catalyzes neither the reduction of O
2
with
reduced rubredoxin nor the reduction of NO with
F
420
H
2
[3]. This difference in reductant specificity is
surprising for homologous enzymes, considering that
F
420
is a deazaflavin (771 Da) that transfers hydride
anions at a redox potential (E°¢)of) 360 mV
[30], whereas rubredoxins are iron–sulfur proteins
(6000 Da) that transfer single electrons at redox poten-
tials around 0 ± 100 mV [31].
We report here on the crystal structures of F
420
H
2
oxidase from Methanothermobacter marburgensis in a
reduced state (2.25 A
˚
) and two oxidized states (1.7 A
˚
and 2.25 A
˚
), and compare them with the 2.5 A
˚

resolu-
tion structure of the rubredoxin:NO ⁄ O
2
oxidoreduc-
tase from Desulfovibrio gigas (31% sequence identity
with F
420
H
2
oxidase) [27] and with the 2.8 A
˚
structure
of the rubredoxin:NO ⁄ O
2
oxidoreductase from Moo-
rella 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 F
420
H
2
oxidase for coenzyme
F
420
H
2
in comparison to that of the two paralogous
enzymes for reduced rubredoxin.
Results and Discussion

Structural basis
F
420
H
2
oxidase from M. marburgensis heterologously
produced in E. coli was isolated and crystallized anaer-
obically and in the presence of dithiothreitol. There-
fore, 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 antiferro-
magnetically coupled [24]. The first structure deter-
mined at 2.25 A
˚
resolution (Table 1) was based on a
crystal in a monoclinic form (grown in the presence of
F
420
H
2
) frozen in liquid nitrogen within the anaerobic
tent. A second and third structure at 2.25 A
˚
and 1.7 A
˚
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)FMNH
2
], 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)FMNH
2
] state super-
imposed. Despite considerable efforts, crystals of the
enzyme were not obtained under aerobic conditions.
F
420
H
2
oxidase from M. marburgensis was found in
the crystals ) according to packing considera-
tions ) as a homotetrameric oligomer (Fig. 2A), which
Fig. 1. Structures of F
420
H
2
and of FMNH
2
, 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 F
420
[52].
H. Seedorf et al. Structure of di-iron flavoenzyme F
420
H
2
oxidase
FEBS Journal 274 (2007) 1588–1599 ª 2007 The Authors Journal compilation ª 2007 FEBS 1589
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 documen-
ted 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 F
420
H
2
oxidase, the inter-
dimer contact areas found in the crystal structures of
rubredoxin:NO ⁄ O
2
oxidoreductase from D. gigas
(7.5%; six ion pairs), and of rubredoxin:NO ⁄ O
2
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 pro-
ductive oligomeric state is the homodimer (see below),

the differences in quaternary structure may reflect dif-
ferences 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 A
˚
between the C
a
atoms of the monomers,
and by the analogous arrangements of the two mono-
mers (Fig. 2B). Briefly, each (F
420
H
2
oxidase) mono-
mer 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 mono-
mer 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 A
˚
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 A
˚
apart, which is too far for
electron transfer at significant rates (Fig. 2).
Table 1. Data collection and refinement statistics
F
420
H
2
oxidase (anaerobic) F
420
H
2
oxidase (air-exposed) F
420
H
2
oxidase (air-exposed)
Crystallization 0.2
M (NH
4
)
2
SO

4
,
0.1
M Mes ⁄ KOH (pH 6.5),
16–22% poly(ethylene glycol)
MME 5000
0.2
M (NH
4
)
2
SO
4
,
0.1
M Mes ⁄ KOH (pH 6.5),
16–22% poly(ethylene glycol)
MME 5000
0.2
M (NH
4
)
2
SO
4
,
0.1
M Mes ⁄ KOH (pH 6.5),
8–16% poly(ethylene glycol)
MME 5000,

15% glycerol
Crystal properties
Space group P2
1
P2
1
P4
3
2
1
2
Cell constants (A
˚
), (°)
No. of monomers in
the asymmetric unit
97.8, 123.1, 135.9, 103.4
8
73.7, 120.9, 92.7, 110.4
4
88.7, 450.4
4
Data collection SLS-X10SA SLS-X10SA SLS-X10SA
Wavelength (A
˚
) 1.000 0.979 0.992
Resolution (A
˚
) 2.25 (2.3–2.25) 1.7 (1.77–1.7) 2.25 (2.32–2.25)
Multiplicity 2.6 (2.5) 4.6 (2.1) 4.5 (2.4)

Completeness (%) 97.6 (97.8) 99.2 (98.9) 97.2 (74.0)
R
sym
(%)
a
6.6 (36.4) 7.8 (41.8) 6.3 (13.7)
I ⁄ r
I
(last shell) 13.7 (1.8) 9.9 (2.3) 16.7 (7.1)
Refinement
R
cryst
(%)
b
20.6 18.6 18.8
R
free
(%)
c
27.0 21.8 23.4
No. of reflections 103 861 156 441 80 298
No. of protein atoms 25 349 12 652 12 652
Average B-factor (A
˚
2
) 44.8, 39.4, 27.4 33.3, 28.9, 20.6 24.8, 20.6, 15.8
Protein, di-iron, FMN
Bond length rms (A
˚
) 0.011 0.018 0.011

Bond angle rms (°) 1.36 1.87 1.30
a
R
sym
¼
P
|I
i
)Ælæ|/
P
I
i
, where I
i
is the observed intensity and Ælæ is the averaged intensity obtained from multiple observations of symmetry-
related reflections.
b
R
cryst
¼
P
hkl
(|F
obs
|)|F
calc
|)/
P
hkl
|F

obs
|.
c
R
cryst
where 5% of the observed reflections (randomly selected) are not used for
refinement.
Structure of di-iron flavoenzyme F
420
H
2
oxidase H. Seedorf et al.
1590 FEBS Journal 274 (2007) 1588–1599 ª 2007 The Authors Journal compilation ª 2007 FEBS
Binding of the di-iron center
The di-iron center differs dramatically between the act-
ive reduced, inactive oxidized and active oxidized
F
420
H
2
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 A
˚
. The described pri-
mary ligation shell essentially corresponds to that
found in the rubredoxin-dependent enzymes. In con-
trast to the latter enzymes, the average site occupancy
of the proximal iron in F
420
H
2
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 air-
exposed tetragonal crystals), the proximal iron is com-
pletely 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 A
˚
,
2.1 A
˚
and 2.5 A
˚
, as well as with the height of the elec-
tron 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 A
˚
, 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 conforma-
tion with respect to the accessibility of the redox cen-
ters 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 ⁄ O
2
oxidoreductase from
D. gigas but not in the 2.8 A
˚
crystal structure of rubre-
doxin:NO ⁄ O
2
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 hyd rogen-bonded to
Arg340 and Lys337 buil d up another new m etal-binding
Fig. 2. Overall structure of F
420
H
2
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 flav-
odoxin-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.
H. Seedorf et al. Structure of di-iron flavoenzyme F
420
H
2
oxidase
FEBS Journal 274 (2007) 1588–1599 ª 2007 The Authors Journal compilation ª 2007 FEBS 1591
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
now hydrogen-bonded to the hydroxyl group of
Ser232, which has also changed its conformation
(Fig. 3B). Notably, a conformational change of a histi-
dine ligated to the distal iron was detected in rubre-
doxin:NO ⁄ O
2
oxidoreductase from D. gigas,in
contrast to the rubredoxin:NO ⁄ O
2
oxidoreductase
from Mo. thermoacetica [28] and F
420
H
2
oxidase.
A third enzyme state was tentatively extracted from
the electron density of the air-exposed monoclinic crys-
tal, which contains both irons in a similar position and
an occupancy as found in the reduced state. Addition-
ally, 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%. Conse-
quently, 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 O
2
reduction. Note that the proximal iron
Fig. 3. Structures of the di-iron-binding site of F
420
H
2
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 glu-

tamate, 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 pep-
tide bond between Leu150 (not shown) and His151. Trp152 shields
the completely buried di-iron center from bulk solvent (monoclinic
crystal resolved to 2.25 A
˚
). (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 A
˚
as part of the switch loop (tetragonal crystal
resolved to 2.25 A
˚
). (C) In the active oxidized state, both the prox-
imal and the distal irons are present as in the active reduced state,
but the switch loop adopts an open conformation (black). The act-
ive 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 A
˚
).
Structure of di-iron flavoenzyme F
420
H
2

oxidase H. Seedorf et al.
1592 FEBS Journal 274 (2007) 1588–1599 ª 2007 The Authors Journal compilation ª 2007 FEBS
is only ligated to Glu85 and Asp170 but not addition-
ally 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 mono-
oxygenase reductase hydroxylase [35] and ribonucleo-
tide reductase [36], where, however, only carboxylate
groups of glutamates and aspartates are subject to
conformational alterations.
O
2
-binding site
The ligand geometry of the di-iron center in the
reduced state offers an attractive O
2
-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 O
2
-bind-
ing pocket is either empty or occupied by a solvent
molecule loosely bound to the distal iron. Whereas in
the inactive oxidized state the O
2
-binding site is des-
troyed, the electron density map derived from the air-

exposed 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 inter-
preted 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
(NH
4
)
2
SO
4
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 sug-
gests 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 monooxyge-
nase 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 hydro-
gen-bonded to His26 and His175.
The shape of the O
2
-binding pocket is approximately
conserved in the structures of rubredoxin:O
2
⁄ NO
oxidoreductases and of F
420
H
2
oxidase, which has no
NO reductase activity. However, the side chains pro-
truding into the pocket partly vary, and might account
for the different specificity. Phe198 in F
420
H
2
oxidase
(Fig. 4) is replaced by tyrosine in the rubredoxin-
dependent enzymes, and the importance of this has
been proven by the decrease of the NO reductase
activity of the Tyr fi Phe mutant in rubredox-
in:NO ⁄ O
2
reductase [28]. Phe198 in F

420
H
2
oxidase
from M. marburgensis is strictly conserved in other
FprA enzymes from methanogenic Archaea (supple-
mentary Fig. S1), most of which contain at least one
FprA with F
420
H
2
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 rubre-
doxin-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
Fig. 4. The O
2
-binding site of F
420
H
2
oxidase. Active site amino
acids, FMN and sulfate are shown as stick models. The dioxygen-
binding 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 interme-
diates (see text). Tyr25 and Phe198 are exchanged in the structur-
ally closely related NO reductases by phenylalanine and tyrosine
(pink). Therefore, Tyr25 and Phe198 are probably responsible for
the finding that F
420
H
2
oxidase does not show NO reductase activ-
ity. 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.
H. Seedorf et al. Structure of di-iron flavoenzyme F
420
H
2
oxidase
FEBS Journal 274 (2007) 1588–1599 ª 2007 The Authors Journal compilation ª 2007 FEBS 1593
der Waals contact with the putative ligand in the
O
2
-binding site, and it might be speculated that its
hydroxyl group interferes with the bulky N
2
O, thus
preventing its formation.
Binding of FMN and modeling of F
420
H

2
The conformation and binding characteristics of FMN
are nearly identical in all of the analyzed structures of
F
420
H
2
oxidase but also in comparison to those of
other members of the FprA family. However, the spe-
cific FMN–polypeptide interactions can be most accu-
rately described in F
420
H
2
oxidase, due to the higher
resolution. FMN has an essentially planar isoalloxa-
zine 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 compo-
nents 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 flavodoxin-
like 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 F
420

H
2
-binding site,
although the experimental verification by structure
determination of an enzyme–F
420
complex was not
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 deazaisoalloxa-
zine ring of F
420
H
2
(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 F
420
H
2
binding.
Model building of F
420
H
2
was governed by the
experimentally determined Si-face stereospecificity of
the hydride transfer to and from F
420

[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 F
420
H
2
and N5 of FMN (Fig. 5), implying that
the generated complex is competent for hydride trans-
fer [40]. Thus positioned, the tricyclic F
420
ring is sand-
wiched 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 F
420
H
2
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 phos-
phodiester portion of F
420
(see Fig. 1) was placed at
the interface between the subunits such that its phos-

phate group is anchored by His117 and His267, which
are both strictly conserved, and its first carboxylate
group by Lys272. In this conformation, the mentioned
F
420
H
2
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 ⁄ O
2
oxidoreductases from D. gigas and of rubredoxin:
NO ⁄ O
2
oxidoreductase from Mo. thermoacetica, the
pocket is filled up from the entrance side by the side
chains of Trp347 and Met146, which are both con-
served in the rubredoxin-dependent enzymes but
replaced by an asparagine and a leucine in F
420
H
2
oxidase (supplementary Figs S1 and S2). F
420
H
2
can-
not 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 F
420
H
2
oxidase is not yet known. The men-
tioned Trp347 would be a candidate for shuttling elec-
trons from rubredoxin to FMN.
The structure-based analysis of the substrate binding
in F
420
H
2
oxidase teaches us once again that, on the
Fig. 5. The F
420
H
2
-binding site of F
420
H
2
oxidase in the active oxid-
ized state. F
420
H
2

(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 F
420
H
2
and N5 of FMN, between which the
hydride is transferred, are positioned within the van der Waals dis-
tance (approximately 3 A
˚
). In this conformation, the Re face of
F
420
H
2
is attached to the switch loop in the open conformation
(black), and the pyrimidine group of F
420
reaches the di-iron center.
Structure of di-iron flavoenzyme F
420
H
2
oxidase H. Seedorf et al.
1594 FEBS Journal 274 (2007) 1588–1599 ª 2007 The Authors Journal compilation ª 2007 FEBS
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 pro-
cess, are strictly conserved. As discussed in detail, only
a few side chain exchanges are sufficient to prevent or
allow NO versus O
2
as electron donor and to block or
favor F
420
H
2
binding over FMN.
The catalytic reaction
The F
420
H
2
oxidase reaction represents a ping-pong
process where, in a first reaction, four electrons from
the diferrous di-iron and FMNH
2
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 F
420
H
2
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)FMNH
2
], for which
the structure has been established. As a first step, the
enzyme binds one molecule of O
2
transiently, forming
a peroxo intermediate bridging the two iron atoms, as
suggested by mechanistic studies with di-iron(II) com-
plexes [41,42]. Then, a first water molecule is released,
leaving behind the enzyme in the diferric l-O(H)
FMNH
2
state (reaction in Scheme 1).
Fe(II)Fe(II)FMNH
2
þ O
2
þ 2H
þ
! Fe(III)OFe(III)FMNH
2
þ H
2
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)FMNH
2
! Fe(III)Fe(III)FMN þ H
2
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 F
420
H
2
and subsequent reduction of FMN, from which the
electrons are shuttled one by one to the irons. After
release of F
420
, a second F
420
H
2
binds, reduces FMN
and leaves the active site (reactions in Schemes 3
and 4).
Fe(III)Fe(III)FMN þ F
420
H
2
! Fe(II)Fe(II)FMN þ F
420

þ 2H
þ
ðScheme 3Þ
Fe(II)Fe(II)FMN þ F
420
H
2
! Fe(II)Fe(II)FMNH
2
þ F
420
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 dime-
thylbenzyl group of FMN and His151 or Asp85
(Fig. 3A). Both residues have a minimal distance to
C8 of the flavin ring of 3.7 A
˚
. 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 pro-
nounced histidines are too far away (4.0–4.5 A

˚
) from
the O
2
-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 O
2
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 O
2.
Structural data suggest
that the sequential course of the complete O
2
reduc-
tion 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 O
2
activa-

tion and block the access of F
420
H
2
(which is compat-
ible with the unsuccessful cocrystallization experiments
with F
420
H
2
oxidase in the reduced state and F
420
H
2
).
When the prosthetic group becomes oxidized upon O
2
reduction, the switch loop is rearranged, thereby abol-
ishing the catalytic competence of the di-iron center
and allowing the binding of F
420
H
2
and the subse-
quent 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 O
2
reduction is real-
ized in a related iron–sulfur and flavin-containing fer-
redoxin oxidase found in methanogenic Archaea, but
H. Seedorf et al. Structure of di-iron flavoenzyme F
420
H
2
oxidase
FEBS Journal 274 (2007) 1588–1599 ª 2007 The Authors Journal compilation ª 2007 FEBS 1595
also in other anaerobic prokaryotes, that catalyze the
reduction of O
2
to H
2
O with H
2
O
2
as free intermedi-
ate [43]. Interestingly, despite its completely different
O
2
activation mechanism, several architectural features
are common to those described for the FprA family,
such as its homotetrameric organization, its head-to-
tail arrangement of two monomers juxtaposing FMN
and the [4Fe)4S] cluster from two different mono-
mers, 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 F
420
H
2
oxidase. However, it is conceivable that the
displacement of the proximal iron to the remote metal-
binding sites over a distance of about 6 A
˚
and 15 A
˚
(Fig. 3B) is related to the inactivation of rubredoxin-
dependent NO reductases after multiple O
2
reduction
cycles. A shift of the proximal iron would be energetic-
ally plausible, as its fixation by ligands is reduced in
the active oxidized state, and because it can move con-
comitantly with the swinging side chain of Glu85 to
constitute, with His26 and His267, an efficient metal-
binding site. Inactivation of FprAs in the presence of
large amounts of O
2
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% N
2
⁄ 5% H
2
(v ⁄ v) and containing a palladium cat-
alyst for O
2
reduction with H
2
. Initial trials to crystallize
F
420
H
2
oxidase were performed with the hanging ⁄ sitting-
drop, 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 solu-
tion (containing 20 mgÆmL
)1
of F
420
H
2
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 (NH
4
)
2
SO
4
,0.1m Mes ⁄ KOH (pH 6.5)
and 30% poly(ethylene glycol) [30% poly(ethylene glycol)
monomethylether 5000] (MME) 5000 or 0.2 m Mg-
formate. 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).
Data collection, structure determination and
refinement
Data were collected at the beam line X10SA of the Swiss-
Light-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 A
˚
structure of rubredoxin:NO ⁄ O
2

oxidoreductase from
Mo. thermoacetica [28]. Using the 2.5 A
˚
structure of rubre-
doxin:NO ⁄ O
2
oxidoreductase from D. gigas [27] gave less
reliable results, although the structures of the two rubre-
doxin-dependent enzymes are very similar, with respect to
both the primary structure (42% sequence identity) and the
quaternary structure (rmsd 1.3 A
˚
for the C
a
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 sep-
arate 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 gener-
ated with pymol (). 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 ()
with accession numbers 2OHI, 2OHH and 2OHJ, respect-
ively.
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.
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H
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oxidase H. Seedorf et al.
1598 FEBS Journal 274 (2007) 1588–1599 ª 2007 The Authors Journal compilation ª 2007 FEBS
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Supplementary material
The following supplementary material is available

online:
Fig. S1. Sequence alignment of F
420
H
2
oxidase from
Methanothermobacter marburgensis, rubredoxin:NO ⁄ O
2
oxidoreductase from Desulfovibrio gigas, rubredoxin:
NO ⁄ O
2
oxidoreductase from Moorella thermoacetica,
F
420
H
2
oxidase from Methanobrevibacter arboriphilus,
and other FprAs from methanogenic archaea assumed
to have F
420
H
2
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-
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 F
420
H
2
pocket at the inter-
face of two subunits in the functional dimer of (A)
F
420
H
2
oxidase from Methanothermobacter marbur-
gensis, (B) rubredoxin:NO ⁄ O
2
oxidoreductase from
Desulfovibrio gigas, and (C) rubredoxin:NO ⁄ O
2
oxido-
reductase from Moorella thermoacetica.
This material is available as part of the online article
from
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 corres-
ponding author for the article.
H. Seedorf et al. Structure of di-iron flavoenzyme F
420
H
2

oxidase
FEBS Journal 274 (2007) 1588–1599 ª 2007 The Authors Journal compilation ª 2007 FEBS 1599