A paramagnetic species with unique EPR characteristics in the active site of heterodisulfide reductase from methanogenic archaea

Heterodisulfide reductase (Hdr) from methanogenic archaea is an iron-sulfur protein that catalyses the reversible reduction of the heterodisulfide (Co M-S-S-CoB) of the methanogenic thiol coenzymes, coenzyme M (H-S-CoM) and co enzyme B (H-S-CoB). In EPR spectroscopic studies with the enzyme from Metha nothermobacter marburgensis, we have identified a unique paramagnetic speci es that is formed upon reaction of the oxidized enzyme with H-S-CoM in the absence of H-S-CoB. This paramagnetic species can be reduced in a one-elect ron step with a midpoint-potential of -185 mV but not further oxidized. A b roadening of the EPR signal in the Fe-57-enriched enzyme indicates that it is at least partially iron based. The g values (g(xyz) = 2.013, 1.991 and 1 .938) and the midpoint potential argue against a conventional [2Fe-2S](+), [3Fe-4S](+), [4Fe-4S](+) or [4Fe-4S](3+) cluster. This species reacts with H-S-CoB to form an EPR silent form. Hence, we propose that only a half reac tion is catalysed in the presence of H-S-CoM and that a reaction intermedia te is trapped. This reaction intermediate is thought to be a [4Fe-4S](3+) c luster that is coordinated by one of the cysteines of a nearby active-site disulfide or by the sulfur of H-S-CoM. A paramagnetic species with similar EPR properties was also identified in Hdr from Methanosarcina barkeri.