HETERODISULFIDE REDUCTASE FROM METHANOL-GROWN CELLS OF METHANOSARCINA-BARKERI IS NOT A FLAVOENZYME

Heterodisulfide reductase from methanol-grown cells of Methanosarcina barkeri (MbHdrDE) is a membrane-bound enzyme composed of a 46-kDa subu nit MbHdrD and a 23-kDa subunit MbHdrE. The enzyme has been shown to c ontain 0.6 mol heme and 20 mol Fe/S per mol heterodimer. in addition, substoichiometric amounts of FAD, thought to be an essential component of the active enzyme, were detected. We have now obtained preparation s of active heterodisulfide reductase in high yields completely devoid of a flavin. Cloning and sequencing of the genes encoding MbHdrD and MbHdrE, which were found to form a transcription unit hdrED, revealed that both subunits also lack an FAD-binding motif. MbHdr thus differs from heterodisulifde reductase from Methanobacterium thermoautotrophic um (MtHdr), which is a flavo iron-sulfur protein composed of the subun its MtHdrA (80 kDa), MtHdrB (36 kDa) and MtHdrC (21 kDa), the subunit HdrA harboring the flavin-binding site. Sequence comparisons revealed that the N-terminal third of MbHdrD, which contained two sequence moti fs for [4Fe-4S] clusters, is similar to MtHdrC and that the C-terminal two thirds of MbHdrD are similar to MtHdrB. Thus, MbHdrD and MtHdrBC are structurally equivalent subunits. MbHdrE shows sequence similarity to b-type cytochromes, in agreement with the finding that this subuni t contains a heme. These and other results indicate that MbHdrD harbor s the active site of heterodisulfide reduction and that a flavin is no t involved in catalysis. Since MbHdrD contains only iron-sulfur cluste rs, a mechanism of disulfide reduction involving one electron rather t han two electron-transfer reactions has to be considered such as opera tive in ferredoxin:thioredoxin reductases from chloroplasts and cyanob acteria.