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: 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 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 (http://www.pymol.org). 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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... 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. .. 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... of hydrogen transfer between NADPH and FEBS Journal 274 (2007) 1588–1599 ª 2007 The Authors Journal compilation ª 2007 FEBS H Seedorf et al 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. .. 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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. 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