Publications

• F420H2 oxidase (FprA) from Methanobrevibacter arboriphilus, a coenzyme F420-dependent enzyme involved in O2 detoxification.

  Seedorf H., Dreisbach A., Hedderich R., Shima S., Thauer R.K.

  Cell suspensions of Methanobrevibacter arboriphilus catalyzed the reduction of O(2) with H(2) at a maximal specific rate of 0.4 U (micromol/min) per mg protein with an apparent K(m) for O(2) of 30 microM. The reaction was not inhibited by cyanide. The oxidase activity was traced back to a coenzyme ... >> More

  Cell suspensions of Methanobrevibacter arboriphilus catalyzed the reduction of O(2) with H(2) at a maximal specific rate of 0.4 U (micromol/min) per mg protein with an apparent K(m) for O(2) of 30 microM. The reaction was not inhibited by cyanide. The oxidase activity was traced back to a coenzyme F(420)-dependent enzyme that was purified to apparent homogeneity and that catalyzed the oxidation of 2 F(420)H(2) with 1 O(2) to 2 F(420) and 2 H(2)O. The apparent K(m) for F(420) was 30 microM and that for O(2) was 2 microM with a V(max) of 240 U/mg at 37 degrees C and pH 7.6, the pH optimum of the oxidase. The enzyme did not use NADH or NADPH as electron donor or H(2)O(2) as electron acceptor and was not inhibited by cyanide. The 45-kDa protein, whose gene was cloned and sequenced, contained 1 FMN per mol and harbored a binuclear iron center as indicated by the sequence motif H-X-E-X-D-X(62)-H-X(18)-D-X(60)-H. Sequence comparisons revealed that the F(420)H(2) oxidase from M. arboriphilus is phylogenetically closely related to FprA from Methanothermobacter marburgensis (71% sequence identity), a 45-kDa flavoprotein of hitherto unknown function, and to A-type flavoproteins from bacteria (30-40%), which all have dioxygen reductase activity. With heterologously produced FprA from M. marburgensis it is shown that this protein is also a highly efficient F(420)H(2) oxidase and that it contains 1 FMN and 2 iron atoms. The presence of F(420)H(2) oxidase in methanogenic archaea may explain why some methanogens, e.g., the Methanobrevibacter species in the termite hindgut, cannot only tolerate but thrive under microoxic conditions. << Less

  Arch. Microbiol. 182:126-137(2004) [PubMed] [EuropePMC]

• Si-face stereospecificity at C5 of coenzyme F420 for F420H2 oxidase from methanogenic Archaea as determined by mass spectrometry.

  Seedorf H., Kahnt J., Pierik A.J., Thauer R.K.

  Coenzyme F420 is a 5-deazaflavin. Upon reduction, 1,5 dihydro-coenzyme F420 is formed with a prochiral centre at C5. All the coenzyme F420-dependent enzymes investigated to date have been shown to be Si-face stereospecific with respect to C5 of the deazaflavin, despite most F420-dependent enzymes ... >> More

  Coenzyme F420 is a 5-deazaflavin. Upon reduction, 1,5 dihydro-coenzyme F420 is formed with a prochiral centre at C5. All the coenzyme F420-dependent enzymes investigated to date have been shown to be Si-face stereospecific with respect to C5 of the deazaflavin, despite most F420-dependent enzymes being unrelated phylogenetically. In this study, we report that the recently discovered F420H2 oxidase from methanogenic Archaea is also Si-face stereospecific. The enzyme was found to catalyse the oxidation of (5S)-[5-2H1]F420H2 with O2 to [5-1H]F420 rather than to [5-2H]F420 as determined by MALDI-TOF MS. (5S)-[5-2H1]F420H2 was generated by stereospecific enzymatic reduction of F420 with (14a-2H2)-[14a-2H2] methylenetetrahydromethanopterin. << Less

  FEBS J. 272:5337-5342(2005) [PubMed] [EuropePMC]

• Structure of coenzyme F420H2 oxidase (FprA), a di-iron flavoprotein from methanogenic Archaea catalyzing the reduction of O2 to H2O.

  Seedorf H., Hagemeier C.H., Shima S., Thauer R.K., Warkentin E., Ermler U.

  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 Meth ... >> More

  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 Methanothermobacter 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 bridging 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-accessible 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. << Less

  FEBS J. 274:1588-1599(2007) [PubMed] [EuropePMC]