We report here on crystal structures of the homotetrameric F420H2 oxidase from Methan-othermobacter marburgensis at resolutions of 2.25 A˚, 2.25 A˚ and 1.7 A˚, respectively, from which a
Trang 1flavoprotein from methanogenic Archaea catalyzing the
Henning Seedorf1, Christoph H Hagemeier1, Seigo Shima1, Rudolf K Thauer1,
Eberhard Warkentin2and Ulrich Ermler2
1 Max Planck Institute for Terrestrial Microbiology, Marburg, Germany
2 Max Planck Institute for Biophysics, Frankfurt am Main, Germany
Oxidases catalyze oxidation reactions with O2 as
elec-tron 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
superox-ide 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-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 F420H2 oxidase that catalyzes a four-electron reduction of O2 to H2O, and have provided evidence that the enzyme is involved in O2 detoxifica-tion in these strictly anaerobic microorganisms [3] In cell extracts of Methanothermobacter thermoautotrophicus,
Keywords
coenzyme F 420 ; crystal structure; di-iron
center; F420H2oxidase; O2detoxification
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 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)FMNH2], 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 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, F420H2was 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 O2 exposure observed
in some members of the FprA family
Abbreviation
F420, 7,8-dimethyl-8-hydroxy-5-deazariboflavin, coenzyme F420.
Trang 2F420H2 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-8-hydroxy-5-deazariboflavin) as electron donor (apparent
Km¼ 30 lm) and O2 as electron acceptor (apparent
Km¼ 2 lm), with an apparent Vmax of the purified
enzyme of 180 s)1[3] Coenzyme F420is a 5-deazaflavin
derivative, and as such transfers hydride anions rather
than single electrons Upon reduction,
1,5-dihydroco-enzyme F420 is formed, with a prochiral center at C5
(Fig 1) The F420H2 oxidase has been shown to be
Si-face stereospecific with respect to C5 of the deazaflavin
[5] Coenzyme F420is 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],
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]
F420H2 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 N2O and
H2O with reduced rubredoxin, but also efficiently
cata-lyzes 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-ter-minus 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
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 F420H2 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⁄ O2 oxidoreduc-tase from Desulfovibrio gigas (31% sequence identity with F420H2oxidase) [27] and with the 2.8 A˚ structure
of the rubredoxin:NO⁄ O2 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 F420H2oxidase 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 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
F420H2) 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 18C 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 super-imposed 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 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 F420[52].
Trang 3is in agreement with previous results (m¼ 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 F420H2 oxidase, the
inter-dimer 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
pro-ductive oligomeric state is the homodimer (see below),
the differences in quaternary structure may reflect
dif-ferences in thermoadaptation rather than difdif-ferences
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 Ca atoms of the monomers,
and by the analogous arrangements of the two
mono-mers (Fig 2B) Briefly, each (F420H2 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 (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 Crystal properties
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
Refinement
Protein, di-iron, FMN
a
R sym ¼ P
|I i )Ælæ|/PI i , where I i is the observed intensity and Ælæ is the averaged intensity obtained from multiple observations of symmetry-related reflections b Rcryst¼ P
hkl (|Fobs| )|F calc |)/ P
hkl |Fobs| c Rcrystwhere 5% of the observed reflections (randomly selected) are not used for refinement.
Trang 4Binding of the di-iron center
The di-iron center differs dramatically between the
act-ive reduced, inactact-ive oxidized and actact-ive 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 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 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 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⁄ O2 oxidoreductase from
D gigasbut not in the 2.8 A˚ crystal structure of rubre-doxin: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
Fig 2 Overall structure of F420H2oxidase (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.
Trang 5site 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⁄ O2 oxidoreductase from D gigas, in contrast to the rubredoxin:NO⁄ O2 oxidoreductase from Mo thermoacetica [28] and F420H2oxidase
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 O2 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 actact-ive reduced state, such that the closed conformation (red) is also visible in the electron density map (monoclinic crystal resolved to 1.7 A ˚ ).
Trang 6is 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
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
-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 O2-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 (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 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 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 pro-truding into the pocket partly vary, and might account for the different specificity Phe198 in F420H2 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 Tyrfi Phe mutant in rubredox-in:NO⁄ O2 reductase [28] Phe198 in F420H2 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 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 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 O2-binding site of F420H2 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 F420H2oxidase 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.
Trang 7der 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
spe-cific FMN–polypeptide interactions can be most
accu-rately described in F420H2 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 F420H2-binding site,
although the experimental verification by structure
determination of an enzyme–F420 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 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 trans-fer [40] Thus positioned, the tricyclic F420ring 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 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 phos-phodiester portion of F420 (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
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 con-served in the rubredoxin-dependent enzymes but replaced by an asparagine and a leucine in F420H2 oxidase (supplementary Figs S1 and S2) F420H2 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 F420H2 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 F420H2 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 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 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 F420reaches the di-iron center.
Trang 8basis 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 O2 as electron donor and to block or
favor F420H2binding 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)
com-plexes [41,42] Then, a first water molecule is released,
leaving behind the enzyme in the diferric l-O(H)
FMNH2state (reaction in Scheme 1)
Fe(II)Fe(II)FMNH2þ O2þ 2 Hþ
! Fe(III)OFe(III)FMNH2þ H2O 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 H2O
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)FMNþ F420H2
! Fe(II)Fe(II)FMN þ F420þ 2 Hþ ðScheme 3Þ
Fe(II)Fe(II)FMNþ F420H2! Fe(II)Fe(II)FMNH2þ 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 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 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 reduc-tion and the complete prosthetic group re-reducreduc-tion 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 activa-tion and block the access of F420H2 (which is compat-ible 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 abol-ishing the catalytic competence of the di-iron center and allowing the binding of F420H2 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 O2 reduction is real-ized in a related iron–sulfur and flavin-containing fer-redoxin oxidase found in methanogenic Archaea, but
Trang 9also in other anaerobic prokaryotes, that catalyze the
reduction of O2 to H2O with H2O2 as free
intermedi-ate [43] Interestingly, despite its completely different
O2activation 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 F420H2 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 O2 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 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
cat-alyst for O2 reduction with H2 Initial trials to crystallize
F420H2 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 F420H2 oxidase) and 2 lL
of reservoir solutions were mixed and incubated at 4C
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
10C 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
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⁄ O2 oxidoreductase from
Mo thermoacetica[28] Using the 2.5 A˚ structure of rubre-doxin:NO⁄ O2 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 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 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 (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, 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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