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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.
