Thiol : fumarate reductase (Tfr) from Methanobacterium thermoautotrophicum

Identification of the catalytic sites for fumarate reduction and thiol oxidation

Authors

  • Steffen Heim,

    1. Max-Planck-Institut für terrestrische Mikrobiologie and Laboratorium für Mikrobiologie des Fachbereichs Biologie der Philipps-Universität, Marburg, Germany
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  • Andreas Künkel,

    1. Max-Planck-Institut für terrestrische Mikrobiologie and Laboratorium für Mikrobiologie des Fachbereichs Biologie der Philipps-Universität, Marburg, Germany
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  • Rudolf K. Thauer,

    1. Max-Planck-Institut für terrestrische Mikrobiologie and Laboratorium für Mikrobiologie des Fachbereichs Biologie der Philipps-Universität, Marburg, Germany
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  • Reiner Hedderich

    1. Max-Planck-Institut für terrestrische Mikrobiologie and Laboratorium für Mikrobiologie des Fachbereichs Biologie der Philipps-Universität, Marburg, Germany
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  • Correspondence to R. Hedderich, Max-Planck-Institut für terrestrische Mikrobiologie, Karl-von-Frisch-Strasse, D-35043 Marburg, Germany

  • Fax: +49 6421 178209.

  • Abbreviations. Tfr, thiol :fumarate reductase; tfr, gene encoding Tfr; Frd, fumarate reductase; Sdh, succinate dehydrogenase; Hdr, heterodisulfide reductase; CoM-S-H, coenzyme M or 2-mercaptoethane sulfonate; CoB-S-H, coenzyme B or 7-mercaptoheptanoylthreonine phosphate; CoM-S-S-CoB, heterodisulfide of CoM-S-H and CoB-S-H; CoM-S-S-CoM, homodisulfide of CoM-S-H; CoB-S-S-CoB, homodisulfide of CoB-S-H.

  • Enzymes. Thiol :fumarate reductase ( EC1.3.4.1); fumarate reductase ( EC1.3.99.1); succinate dehydrogenase ( EC1.3.5.1); heterodisulfide reductase (EC 1.99.4.−); methyl-coenzyme M reductase (EC 1.8.99.−).

  • Note. The DNA sequences published here have been submitted to the EMBL sequence data bank and are available under accession number TfrA, AJ 000941; TfrB, AJ 000942.

Abstract

Most methanogenic Archaea contain an unusual cytoplasmic fumarate reductase which catalyzes the reduction of fumarate with coenzyme M (CoM-S-H) and coenzyme B (CoB-S-H) as electron donors forming succinate and CoM-S-S-CoB as products. We report here on the purification and characterization of this thiol :fumarate reductase (Tfr) from Methanobacterium thermoautotrophicum (strain Marburg). The purified enzyme, which was composed of two different subunits with apparent molecular masses of 58 kDa (TfrA) and 50 kDa (TfrB), was found to catalyze the following reactions : (a) the reduction of fumarate with CoM-S-H and CoB-S-H (150 U/mg); (b) the reduction of fumarate with reduced benzyl viologen (620 U/mg); (c) the oxidation of CoM-S-H and CoB-S-H to CoM-S-S-CoB with methylene blue (95 U/mg); and (d) the reduction of CoM-S-S-CoB with reduced benzyl viologen (250 U/mg). The flavoprotein contained 12 mol non-heme iron and approximately the same amount of acid-labile sulfur/mol heterodimer. The genes encoding TfrA and TfrB were cloned and sequenced. Sequence comparisons with fumarate reductases and succinate dehydrogenases from Bacteria and Eucarya and with heterodisulfide reductases from M. thermoautotrophicum and Methanosarcina barkeri revealed that TfrA harbors FAD-binding motifs and the catalytic site for fumarate reduction and that TfrB harbors one [2Fe-2S] cluster and two [4Fe-4S] clusters and the catalytic site for CoM-S-H and CoB-S-H oxidation.

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