Si-face stereospecificity at C5 of coenzyme F 420 for F 420 H 2 oxidase from methanogenic Archaea as determined by mass spectrometry Henning Seedorf 1 ,Jo ¨ rg Kahnt 1 , Antonio J. Pierik 2 and Rudolf K. Thauer 1 1 Max Planck Institute for Terrestrial Microbiology, Marburg, Germany 2 Fachbereich Biologie, Philipps-Universita ¨ t, Marburg, Germany Methanogenic Archaea fluoresce greenish yellow when irradiated with UVA light. The fluorescence is due to coenzyme F 420 , a 7,8-didemethyl-8-hydroxy-5-deaza- riboflavin derivative (Fig. 1). The coenzyme, which is generally present in 1 mm intracellular concentrations [1], functions as a redox mediator in methanogenesis, in NADP + reduction and in glucose-6-phosphate dehydrogenation [2]. With respect to its redox proper- ties, F 420 is much more similar to pyridine nucleotides than to flavins [3]. Both F 420 and NAD(P) transfer hydride anions and not single electrons. In the reduced form, both F 420 and NAD(P) have a prochiral centre, F 420 at C5 and NAD(P) at C4. They differ function- ally, mainly in that the redox potential of the F 420 ⁄ F 420 H 2 pair (E°¢ ¼ )360 mV) is 40 mV more negative than that of the NAD(P) + ⁄ NAD(P)H pair (E°¢ ¼ )320 mV) [5]. All F 420 -dependent enzymes analysed to date in this respect have been shown to be Si-face stereospecific at C5 of F 420 [6]. This is surprising because NAD(P)- dependent enzymes can be Si-face or Re-face specific [7] and some flavoenzymes, whose apoproteins catalyse the reduction of synthetic 8-hydroxy-5-deaza-FAD, have been shown to be Si-face stereospecific with respect to C5 of the synthetic deazaflavin and others to be Re-face stereospecific [7,8]. In case of the pyridine-nucleotide-dependent enzymes the redox potential (E°) of the electron acceptor reduced by NAD(P)H is thought to be an important factor deter- mining the stereospecificity of these enzymes [9,10]. Keywords coenzyme F 420 ; 5-deazaflavins; F 420 H 2 oxidase; methanogenic Archaea; stereospecificity Correspondence R. K. Thauer, Max Planck-Institute for Terrestrial Microbiology, Karl-von-Frisch- Strasse, D-35043 Marburg, Germany Fax: +49 642 117 8109 Tel: +49 642 117 8101 E-mail: thauer@staff.uni-marburg.de (Received 4 July 2005, revised 17 August 2005, accepted 23 August 2005) doi:10.1111/j.1742-4658.2005.04931.x Coenzyme F 420 is a 5-deazaflavin. Upon reduction, 1,5 dihydro-coenzyme F 420 is formed with a prochiral centre at C5. All the coenzyme F 420 -dependent enzymes investigated to date have been shown to be Si-face stereospecific with respect to C5 of the deazaflavin, despite most F 420 - dependent enzymes being unrelated phylogenetically. In this study, we report that the recently discovered F 420 H 2 oxidase from methanogenic Archaea is also Si-face stereospecific. The enzyme was found to catalyse the oxidation of (5S)-[5- 2 H 1 ]F 420 H 2 with O 2 to [5- 1 H]F 420 rather than to [5- 2 H]F 420 as determined by MALDI-TOF MS. (5S)-[5- 2 H 1 ]F 420 H 2 was generated by stereospecific enzymatic reduction of F 420 with (14a- 2 H 2 )- [14a- 2 H 2 ] methylenetetrahydromethanopterin. Abbreviations Adf, F 420 -specific alcohol dehydrogenase; F 420 , coenzyme F 420 ; Fgd, F 420 -dependent glucose-6-phosphate dehydrogenase; Fno, F 420 H 2 : NADP + oxidoreductase; Fpo, F 420 H 2 dehydrogenase complex; FprA, F 420 H 2 oxidase; Frd, F 420 -dependent formate dehydrogenase; Frh, F 420 -reducing hydrogenase; H 4 MPT, tetrahydromethanopterin; Mer, F 420 -dependent methylenetetrahydromethanopterin reductase; methylene-H 4 MPT, methylenetetrahydromethanopterin; Mtd, F 420 -dependent methylenetetrahydromethanopterin dehydrogenase; TFA, trifluoroacetic acid. FEBS Journal 272 (2005) 5337–5342 ª 2005 FEBS 5337 Redox potentials (E°) of the electron acceptor more neg- ative than )200 mV generally promote Si-face stereo- specificity and redox potentials more positive than )200 mV promote Re-face stereospecificity at C4 of NAD(P). Thus the NADP-dependent glucose-6-phos- phate dehydrogenase is Si-face specific (E° ¼ )330 mV) and the NAD-dependent malate dehydro- genase is Re-face specific (E° ¼ )170 mV). Most eth- anol dehydrogenases are Re-face specific (E° ¼ 200 mV), but the enzyme from Drosophila melanogaster is Si-face specific. For recent literature on the subject see Berk et al. [11]. The following eight F 420 -dependent enzymes have been analysed and shown to be Si-face specific: F 420 - reducing hydrogenase (Frh) [12,13]; F 420 -dependent formate dehydrogenase (Frd) [14]; F 420 -specific alcohol dehydrogenase (Adf) [6]; F 420 -dependent methylene- tetrahydromethanopterin dehydrogenase (Mtd) [15,16]; F 420 -dependent methylenetetrahydromethanopterin reductase (Mer) [15]; F 420 H 2 dehydrogenase complex (Fpo) [15]; F 420 H 2 : NADP + oxidoreductase (Fno) [17,18]; and F 420 -dependent glucose-6-phosphate de- hydrogenase (Fgd) [6]. Adf, Mer and Fgd form a fam- ily, as do the F 420 -binding subunits FpoF, FrhB and FrdB. The two families, Mtd and Fno are not phylo- genetically related. The eight enzymes catalyse redox reactions with electron acceptors ranging in redox potential (E° ¢ ) from )414 mV (2H + ⁄ H 2 )to)165 mV (methanophenazine ox ⁄ red) [19]. The Si-face stereospe- cificity of F 420 -dependent enzymes thus appears to be independent of their phylogenetic origin and of the thermodynamics of the reactions catalysed by them. Recently a novel F 420 -dependent enzyme, F 420 H 2 oxidase (FprA), was discovered in methanogenic Arch- aea [20]. FprA catalyses the oxidation of 2 F 420 H 2 with O 2 to 2 F 420 and 2 H 2 O. The 45 kDa protein contains 1 FMN per mol and harbours a binuclear iron centre indicated by the sequence motif H-X-E-X-D-X 63 -H- X 18 -D-X 62 -H. FprA is not phylogenetically related to any of the other F 420 -dependent enzymes and cata- lyses a reaction with a redox potential difference of +1.27 V (F 420 H 2 oxidation with O 2 ). We therefore investigated the stereospecificity of this enzyme and found it to be Si-face specific at C5 of F 420 . The method used was based, in principle, on the technique for determining the hydride transfer stereospecificity of nicotinamide adenine dinucleotide-linked oxidoreduc- tases by MS [21]. Results The following findings are important for the under- standing of the results shown in Fig. 2: (a) [5- 1 H]F 420 and [5- 2 H]F 420 can be identified and the relative amounts present in a mixture quantitated using MALDI-TOF-MS; (b) F 420 H 2 auto-oxidizes nonstereo- specifically to F 420 in the matrix used for MALDI- TOF-MS; (c) F 420 is stereospecifically reduced to F 420 H 2 with methylenetetrahydromethanopterin (methylene-H 4 MPT) in the presence of F 420 -dependent methylene-H 4 MPT dehydrogenase (Mtd), which has been shown to be Si-face specific at C5 of F 420 [15,16]; and (d) F 420 is chemically reduced to F 420 H 2 with NaBH 4 in a nonstereospecific reaction. In Fig. 2A three MALDI-TOF mass spectra of a control experiment are shown: the spectrum of [5- 1 H]F 420 (Fig. 2Aa); the spectrum of [5- 1 H 2 ]F 420 H 2 generated from [5- 1 H]F 420 by Mtd-catalysed reduction with [14a- 1 H 2 ]methylene-H 4 MPT (Fig. 2Ab); and the spectrum of [5- 1 H]F 420 generated from [5- 1 H 2 ]F 420 H 2 by FprA catalysed oxidation (Fig. 2Ac). As seen from the normalized 1 Da separated stick spectra (insets, black) all three mass spectra are almost identical to the stick spectrum (insets, white) calculated for [5- 1 H]F 420 from its elemental composition considering the isotope composition of the elements: 98.9% 12 C, 1.1% 13 C; 99.63% 14 N, 0.37% 15 N; 99.99% 1 H, 0.01% 2 H; and 99.76% 16 O, 0.24% 17 O and 18 O. The experiment shown in Fig. 2B differs from that in Fig. 2A only in that in the first step F 420 was enzy- matically reduced with [14a- 2 H 2 ] methylene-H 4 MPT yielding (5S)-[5- 2 H 1 ]F 420 H 2 . FprA catalysed oxidation of (5S)-[5- 2 H 1 ]F 420 H 2 yielded only[5– 1 H]F 420 as indica- ted by the mass spectrum (Fig. 2Bc), which was identi- cal to that calculated for [5- 1 H]F 420 (Fig. 2Ba). This result can only be explained if FprA is Si-face specific with respect to C5 of F 420 . In contrast, auto-oxidation of (5S)-[5- 2 H 1 ]F 420 H 2 yielded a 1 : 2 mixture of [5- 1 H]F 420 and [5- 2 H]F 420 , as indicated by the relative intensities of the 772 and 773 Da mass peaks Fig. 1. Structure of reduced coenzyme F 420 (F 420 H 2 ). F 420 ¼ N-(N- L-lactyl-L-glutamyl)-L-glutamic acid phosphodiester of 7,8-didemethyl- 8-hydroxy-5-deazariboflavin. Stereochemistry of F 420 H 2 oxidase H. Seedorf et al. 5338 FEBS Journal 272 (2005) 5337–5342 ª 2005 FEBS (Fig. 2Bb, stick spectrum, black). For comparison the relative intensities calculated for a 1 : 1 mixture are given (Fig. 2Bb, stick spectrum, white). The [5- 1 H]F 420 to [5- 2 H]F 420 ratio of 1 : 2 can be explained assuming a deuterium isotope effect of  2 for the auto-oxida- tion reaction. As a control, F 420 was reduced with NaB 2 H 4 (NaBD 4 ) yielding a mixture of (5S)-[5- 2 H 1 ]F 420 H 2 and (5R)-[5- 2 H 1 ]F 420 H 2 . The FprA-catalysed oxidation of the mixture yielded a 1 : 1 mixture of [5- 1 H]F 420 and [5- 2 H]F 420 as revealed by the relative intensities of the 772 and 773 Da mass peaks (Fig. 2Cc). The results are consistent with FprA catalyzing the oxidation of (5S)- [5- 2 H 1 ]F 420 H 2 to [5- 1 H]F 420 and the oxidation of (5R)- [5- 2 H 1 ]F 420 H 2 to [5- 2 H]F 420 as to be expected for a Si-face-specific enzyme. The finding that the 775 Da mass peak in Fig. 2Cb was much lower than in Fig. 2Bb is probably due to the fact that reduction of F 420 with NaBD 4 (Fig. 2C) was not complete and therefore after auto-oxidation the F 420 analysed contained less 2 H. Discussion In the Introduction it was pointed out that all F 420 - dependent enzymes investigated have been shown to be Si-face specific at C5 of F 420 , despite four of these enzymes being unrelated phylogenetically. The finding that F 420 H 2 oxidase (FprA) is also Si-face specific brings to five the number of Si-face-specific F 420 -dependent enzymes that are not related phylogenetically. There is only a 6.25% probability that this is by chance. To date, the crystal structures of four F 420 -dependent enzymes have been resolved: F 420 H 2 :NADP oxido- reductase, with and without F 420 bound [22]; F 420 -dependent alcohol dehydrogenase with F 420 bound [23]; Mer, with and without F 420 bound [24,25]; and Mtd without F 420 bound [26]. A common F 420 -binding Fig. 2. MALDI-TOF-MS analysis of F 420 and F 420 H 2 for the deter- mination of the stereospecificity of F 420 H 2 oxidase (FprA). The insets show normalized 1 Da separated stick spectra obtained from the measured data (black) aligned to simulated spectra (white). For better visibility in the structures deuterium is abbreviated by D rather than by 2 H and hydrogen by H rather than 1 H. Mtd, Si-face- specific F 420 -dependent methylenetetrahydromethanopterin de- hydrogenase. (A) Experiment with nonlabelled substrates showing that the mass spectrum of [5- 1 H 2 ]F 420 H 2 (b), owing to auto-oxida- tion of F 420 H 2 , is identical to that of [5- 1 H]F 420 (a, c). The simulated stick spectra (white) are for [5- 1 H]F 420 . (B) Experiment with specif- ically 2 H-labelled substrates showing that the mass spectrum of F 420 formed from (5S)-[5- 2 H 1 ]F 420 H 2 by FprA-catalysed oxidation (c) is identical to the spectrum of [5- 1 H]F 420 (a). The simulated stick spectrum (b, white) of (5S)-[5- 2 H 1 ]F 420 H 2 is for a 1 : 1 mixture of [5- 1 H]F 420 and [5- 2 H]F 420 . The other two (a, c) are for [5- 1 H]F 420 .(C) Experiment with NaB 2 H 4 -reduced [5- 1 H]F 420 showing that the mass spectrum of F 420 formed from reduced F 420 by FprA-catalysed oxi- dation corresponds to that of a mixture of [5- 1 H]F 420 and [5- 2 H]F 420 (c). The simulated stick spectrum of reduced F 420 (b, white) and that of the FprA oxidation product (c, white) are for a 1 : 1 mixture of [5- 1 H]F 420 and [5- 2 H]F 420 . H. Seedorf et al. Stereochemistry of F 420 H 2 oxidase FEBS Journal 272 (2005) 5337–5342 ª 2005 FEBS 5339 motif explaining the Si-face specificity of these enzymes was not found. It therefore has to be considered that Si-face specificity may be an intrinsic property of F 420 rather than of the F 420 -dependent enzymes. An example of the stereospecificity of a dehydrogenase being dicta- ted by the structure of its substrate has recently been published. There are several phylogenetically unrelated methylenetetrahydromethanopterin dehydrogenases and methylenetetrahydrofolate dehydrogenases that are all Re-face specific at the carbon of the methylene group [27,28]. It has been calculated that the transition state conformation of methylenetetrahydromethanopterin and methylenetetrahydrofolate for the dehydrogenation from the Re-face is energetically favoured [28]. How- ever, in the case of F 420 , there are no centres of asym- metry in the near neighbourhood of C5 that could interact with the reactant or the product, or affect the transition state(s) and by that induce an intrinsic ener- getic difference in the reaction profiles involving the Si versus the Re side of F 420 . The nearest asymmetry cen- tres are in the N10 side chain. It is therefore difficult to envisage how the Si-face stereospecificity of F 420 - dependent enzymes could be dictated by the structure of F 420 . Experimental procedures Isotopes, coenzymes and enzymes Deuterium oxide ( 2 H 2 O) and deuterated formaldehyde ( 2 H 2 CO) were from Euriso-Top (Saarbru ¨ cken, Germany) and sodium borodeuteride (NaB 2 H 4 ) was from Fluka (Tauf- kirchen, Germany). Coenzyme F 420 and tetrahydrometha- nopterin (H 4 MPT) were purified from Methanothermobacter marburgensis (DSMZ 2133) [29] [14a- 1 H 2 ]methylene-H 4 MPT was prepared by spontaneous reaction of H 4 MPT and 1 H 2 CO and [14a- 2 H 2 ]methylene-H 4 MPT by spontaneous reaction of H 4 MPT and 2 H 2 CO [30]. FprA from M. marbur- gensis [20] and Mtd from Methanopyrus kandleri [26] were produced heterologously in Escherichia coli and purified to specific activities of 100 and 4000 UÆmg )1 , respectively (1 U ¼ 1 lmolÆmin )1 ). Protein was determined with the Rot- Nanoquant-Microassay from Roth (Karlsruhe, Germany) using bovine serum albumin as standard. Assay to determine the stereospecificity of F 420 H 2 oxidase The assay is described in Fig. 2B. The 1.2 mL assay mix- ture at 30 °C contained 60 lm H 4 MPT, 140 lm 2 H 2 CO and 55 lm F 420 in oxic 120 mm potassium phosphate pH 6. Reduction of F 420 to (5S)-[ 2 H 1 ]F 420 H 2 with [14a- 2 H 2 ]methy- lene-H 4 MPT (spontaneously generated from H 4 MPT and 2 H 2 CO) was started by the addition of 120 U Mtd (Si-face specific) and was completed after 5 min. Subsequently, 60 U FprA were added, which catalysed the oxidation of F 420 H 2 with O 2 as the electron acceptor. Samples of the assay were taken before and 5 min after the addition of Mtd and 5 min after the addition of FprA and analysed by MALDI-TOF-MS. In the control experiment described in Fig. 2A, the 1.2 mL assay mixture contained 140 lm 1 H 2 CO instead of 140 lm 2 H 2 CO. In the control experiment described in Fig. 2C, the 1.2 mL assay did not contain H 4 MPT, H 2 CO or Mtd. Instead, F 420 was reduced with NaB 2 H 4 to a mixture of (5S)-[5- 2 H 1 ]F 420 H 2 and (5R)-[5- 2 H 1 ]F 420 H 2 . This step was carried out under anaerobic conditions. Analysis of F 420 and F 420 H 2 by MALDI-TOF-MS Samples (25 lL) of the 1.2 mL assay mixtures were applied to a small ZipTips (Millipore Corp, Bedford, MA, USA) column previously equilibrated with 0.1% (v ⁄ v) trifluoro- acetic acid (TFA). The column was then washed with 0.1% (v ⁄ v) TFA to remove salts and was then eluted with 84% (v ⁄ v) acetonitrile ⁄ 0.1% (v ⁄ v) TFA. The eluate was dried by vacuum centrifugation and the dried pellet dissolved in 10 lL 0.1% (v ⁄ v) TFA and subsequently supplemented with 10 lL of a saturated solution of a -cyano-4-hydroxy- cinnamic acid in 70% (v ⁄ v) acetonitrile ⁄ 0.1% (v ⁄ v) TFA. Aliquots were air dried and analysed by MALDI-TOF-MS. The mass spectra were collected in the reflector negative- ion mode. For each spectrum, at least 150 single shots were summed. The spectra were determined with a Voyager DE RP from PE Biosystems. The natural isotopic distribution in F 420 was calculated by the isotope pattern calculator provided by the University of Sheffield at the ChemPuter site (http://www.shef.ac.uk/ chemistry/chemputer/). All calculations of simulated data were carried out in excel 2000 and transformed into stick spectra separated by 1 Da [31]. Acknowledgements This work was supported by the Max Planck Society and by the Fonds der Chemischen Industrie. Henning Seedorf thanks the Deutsche Forschungsgemeinschaft for a graduate fellowship. We are indebted to Christ- oph Hagemeier for providing purified F 420 -dependent Mtd from Methanopyrus kandleri. References 1 Scho ¨ nheit P, Keweloh H & Thauer RK (1981) Factor F 420 degradation in Methanobacterium thermoauto- Stereochemistry of F 420 H 2 oxidase H. Seedorf et al. 5340 FEBS Journal 272 (2005) 5337–5342 ª 2005 FEBS trophicum during exposure to oxygen. FEMS Microbiol Lett 12, 347–349. 2 Thauer RK (1998) Biochemistry of methanogenesis: a tribute to Marjory Stephenson. Microbiology 144, 2377– 2406. 3 Jacobson F & Walsh C (1984) Properties of 7,8-di- demethyl-8-hydroxy-5-deazaflavins relevant to redox coenzyme function in methanogen metabolism. Biochemistry 23, 979–988. 4 Walsh C (1986) Naturally occurring 5-deazaflavin coenzymes: biological redox roles. Acc Chem Res 19, 216–221. 5 Gloss LM & Hausinger RP (1987) Reduction potential characterization of methanogen factor 390. FEMS Microbiol Lett 48, 143–145. 6 Klein AR, Berk H, Purwantini E, Daniels L & Thauer RK (1996) Si-face stereospecificity at C5 of coenzyme F 420 for F 420 -dependent glucose-6-phosphate dehydro- genase from Mycobacterium smegmatis and F 420 -depen- dent alcohol dehydrogenase from Methanoculleus thermophilicus. Eur J Biochem 239, 93–97. 7 Creighton DJ & Murthy NSRK (1990) Stereochemistry of enzyme-catalyzed reactions at carbon. In The Enzymes (Sigman DS & Boyer PD, eds), Vol. XIX, pp. 323–421. Academic Press, San Diego. 8 Sumner JS & Matthews RG (1992) Stereochemistry and mechanism of hydrogen transfer between NADPH and methylenetetrahydrofolate in the reaction catalyzed by methylenetetrahydrofolate reductase from pig liver. J Am Chem Soc 114, 6949–6956. 9 Benner SA (1982) The stereoselectivity of alcohol dehydrogenases: a stereochemical imperative? Experientia 38, 633–637. 10 Nambiar KP, Stauffer DM, Kolodziej PA & Benner SA (1983) A mechanistic basis for the stereoselectivity of enzymatic transfer of hydrogen from nicotinamide cofactors. J Am Chem Soc 105, 5886–5890. 11 Berk H, Buckel W, Thauer RK & Frey PA (1996) Re-face stereospecificity at C4 of NAD(P) for alcohol dehydrogenase from Methanogenium organophilum and for (R)-2-hydroxyglutarate dehydrogenase from Acidaminococcus fermentans as determined by 1 H-NMR spectroscopy. FEBS Lett 399, 92–94. 12 Teshima T, Nakai S, Shiba T, Tsai L & Yamazaki J (1985) Elucidation of stereospecificity of a selenium- containig hydrogenase from Methanococcus vannielii – syntheses of (R)- and S-[4-H1]-3,4-dihydro-7-hydroxy- 1-hydroxyethylquinolinone. Tetrahedron Lett 26, 351–354. 13 Yamazaki S, Tsai L, Stadtman TC, Teshima T, Nakaji A & Shiba T (1985) Stereochemical studies of a selenium-containing hydrogenase from Methanococcus vannielii: determination of the absolute configuration of C-5 chirally labeled dihydro-8-hydroxy-5-deazaflavin cofactor. Proc Natl Acad Sci USA 82, 1364–1366. 14 Schauer NL, Ferry JG, Honek JF, Orme-Johnson WH & Walsh C (1986) Mechanistic studies of the coenzyme F 420 reducing formate dehydrogenase from Methano- bacterium formicicum. Biochemistry 25, 7163–7168. 15 Kunow J, Schwo ¨ rer B, Setzke E & Thauer RK (1993) Si- face stereospecificity at C5 of coenzyme F 420 for F 420 - dependent N5,N10-methylenetetrahydromethanopterin dehydrogenase, F 420 -dependent N5,N10–methylene- tetrahydromethanopterin reductase and F 420 H 2 : dimeth- ylnaphthoquinone oxidoreductase. Eur J Biochem 214, 641–646. 16 Klein AR & Thauer RK (1995) Re-face specificity at C14a of methylenetetrahydromethanopterin and Si-face specificity at C5 of coenzyme F 420 for coenzyme F 420 - dependent methylenetetrahydromethanopterin dehydro- genase from methanogenic Archaea. Eur J Biochem 227, 169–174. 17 Yamazaki S, Tsai L, Stadtman TC, Jacobson FS & Walsh C (1980) Stereochemical studies of 8-hydroxy- 5-deazaflavin-dependent NADP + reductase from Methanococcus vannielii. J Biol Chem 255, 9025– 9027. 18 Kunow J, Schwo ¨ rer B, Stetter KO & Thauer RK (1993) AF 420 -dependent NADP reductase in the extremely thermophilic sulfate-reducing Archaeoglobus fulgidus. Arch Microbiol 160, 199–205. 19 Tietze M, Beuchle A, Lamla I, Orth N, Dehler M, Greiner G & Beifuss U (2003) Redox potentials of methanophenazine and CoB-S-S-CoM, factors involved in electron transport in methanogenic Archaea. Chem- biochem 4, 333–335. 20 Seedorf H, Dreisbach A, Hedderich R, Shima S & Thauer RK (2004) F 420 H 2 oxidase (FprA) from Methanobrevi- bacter arboriphilus, a coenzyme F 420 -dependent enzyme involved in O 2 detoxification. Arch Microbiol 182, 126– 137. 21 Ehmke A, Flossdorf J, Habicht W, Schiebel HM & Schulten HR (1980) A new technique for determining the hydride transfer stereospecificity of nicotinamide adenine dinucleotide-linked oxidoreductases: electron impact and field desorption mass spectrometry. Anal Biochem 101, 413–420. 22 Warkentin E, Mamat B, Sordel-Klippert M, Wicke M, Thauer RK, Iwata M, Iwata S, Ermler U & Shima S (2001) Structures of F 420 H 2 : NADP + oxidoreductase with and without its substrates bound. EMBO J 20, 6561–6569. 23 Aufhammer SW, Warkentin E, Berk H, Shima S, Thauer RK & Ermler U (2004) Coenzyme binding in F 420 -dependent secondary alcohol dehydrogenase, a member of the bacterial luciferase family. Structure 12, 361–370. 24 Shima S, Warkentin E, Grabarse W, Sordel M, Wicke M, Thauer RK & Ermler U (2000) Structure of coen- zyme F 420 dependent methylenetetrahydromethanopterin H. Seedorf et al. Stereochemistry of F 420 H 2 oxidase FEBS Journal 272 (2005) 5337–5342 ª 2005 FEBS 5341 reductase from two methanogenic archaea. J Mol Biol 300, 935–950. 25 Aufhammer SW, Warkentin E, Ermler U, Hagemeier CH, Thauer RK & Shima S (2005) Crystal structure of methylenetetrahydromethanopterin reductase (Mer) in complex with coenzyme F 420 : architecture of the F 420 ⁄ FMN binding site of enzymes within the nonprolyl cis-peptide containing bacterial luciferase family. Protein Sci 14, 1840–1849. 26 Hagemeier CH, Shima S, Thauer RK, Bourenkov G, Bartunik HD & Ermler U (2003) Coenzyme F 420 -depen- dent methylenetetrahydromethanopterin dehydrogenase (Mtd) from Methanopyrus kandleri: a methanogenic enzyme with an unusual quarternary structure. J Mol Biol 332, 1047–1057. 27 Vorholt JA, Chistoserdova L, Lidstrom ME & Thauer RK (1998) The NADP-dependent methylene tetrahydro- methanopterin dehydrogenase in Methylobacterium extorquens AM1. J Bacteriol 180 , 5351–5356. 28 Bartoschek S, Buurman G, Thauer RK, Geierstanger BH, Weyrauch JP, Griesinger C, Nilges M, Hutter MC & Helms V (2001) Re-face stereospecificity of methylenetetrahydromethanopterin and methylenetetrahydrofolate dehydrogenases is predeter- mined by intrinsic properties of the substrate. Chembio- chem 2, 530–541. 29 Shima S & Thauer RK (2001) Tetrahydromethanop- terin-specific enzymes from Methanopyrus kandleri. Methods Enzymol 331, 317–353. 30 Escalante-Semerena JC, Rinehart KL Jr & Wolfe RS (1984) Tetrahydromethanopterin, a carbon carrier in methanogenesis. J Biol Chem 259, 9447–9455. 31 Selmer T, Kahnt J, Goubeaud M, Shima S, Grabarse W, Ermler U & Thauer RK (2000) The biosynthesis of methylated amino acids in the active site region of methyl-coenzyme M reductase. J Biol Chem 275, 3755– 3760. Stereochemistry of F 420 H 2 oxidase H. Seedorf et al. 5342 FEBS Journal 272 (2005) 5337–5342 ª 2005 FEBS . Si-face stereospecificity at C5 of coenzyme F 420 for F 420 H 2 oxidase from methanogenic Archaea as determined by mass spectrometry Henning. [5- 1 H]F 420 showing that the mass spectrum of F 420 formed from reduced F 420 by FprA-catalysed oxi- dation corresponds to that of a mixture of [5- 1 H]F 420 and