Heterodisulfide Reductase from Methanol‐Grown Cells of Methanosarcina Barkeri is not a Flavoenzyme

Heterodisulfide reductase from methanol‐grown cells of Methanosarcina barkeri ( Mb HdrDE) is a membrane‐bound enzyme composed of a 46‐kDa subunit Mb HdrD and a 23‐kDa subunit Mb HdrE. The enzyme has been shown to contain 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 preparations of active heterodisulfide reductase in high yields completely devoid of a flavin. Cloning and sequencing of the genes encoding Mb HdrD and Mb HdrE, which were found to form a transcription unit hdrED revealed that both subunits also lack an FAD‐binding motif. Mb Hdr thus differs from heterodisulifde reductase from Methanobacterium thermoautotrophicum ( Mt Hdr), which is a flavo iron‐sulfur protein composed of the subunits Mt HdrA (80 kDa), Mt HdrB (36 kDa) and Mt HdrC (21 kDa), the subunit HdrA harboring the flavin‐binding site. Sequence comparisons revealed that the N‐terminal third of Mb HdrD, which contained two sequence motifs for [4Fe‐4S] clusters, is similar to Mt HdrC and that the C‐terminal two thirds of Mb HdrD are similar to Mt HdrB. Thus, Mb HdrD and Mt HdrBC are structurally equivalent subunits. Mb HdrE shows sequence similarity to b ‐type cytochromes, in agreement with the finding that this subunit contains a heme. These and other results indicate that Mb HdrD harbors the active site of heterodisulfide reduction and that a flavin is not involved in catalysis. Since Mb HdrD contains only iron‐sulfur clusters, a mechanism of disulfide reduction involving one electron rather than two electron‐transfer reactions has to be considered such as operative in ferredoxin:thioredoxin reductases from chloroplasts and cyanobacteria.