Evidence for the involvement of two heterodisulfide reductases in the energy‐conserving system of Methanomassiliicoccus luminyensis

Methanomassiliicoccus luminyensis was isolated from the human gut, and requires H 2 and methanol or methylamines to produce methane. The organism lacks cytochromes, indicating that it cannot couple membrane‐bound electron transfer reactions with extrusion of H + or Na + ions using known methanogenic pathways. Furthermore, M . luminyensis contains a soluble hydrogenase/heterodisulfide reductase complex (Mvh AGD /Hdr ABC ) as found in obligate hydrogenotrophic methanogens, but the energy‐conserving methyltransferase (MtrA‐H) is absent. Thus, the question arises as to how this species synthesizes ATP . We present evidence that M. luminyensis uses two types of heterodisulfide reductases (Hdr ABC and HdrD) in a novel process for energy conservation. Quantitative RT ‐ PCR studies revealed that genes encoding these heterodisulfide reductases showed high expression levels. Other genes with high transcript abundance were fpoA (part of the operon encoding the ‘headless’ F 420 H 2 dehydrogenase) and atpB (part of the operon encoding the A 1 A o ATP synthase). High activities of the soluble heterodisulfide reductase Hdr ABC and the hydrogenase Mvh ADG were found in the cytoplasm of M. luminyensis . Also, heterologously produced HdrD was able to reduce CoM‐S‐S‐CoB using reduced methylviologen as an electron donor. We propose that membrane‐bound electron transfer is based on conversion of two molecules of methanol and concurrent formation of two molecules of the heterodisulfide CoM‐S‐S‐CoB. First the Hdr ABC /Mvh ADG complex catalyzes the H 2 ‐dependent reduction of CoM‐S‐S‐CoB and formation of reduced ferredoxin. In a second cycle, reduced ferredoxin is oxidized by the ‘headless’ F 420 H 2 dehydrogenase, thereby translocating up to 4 H + across the membrane, and electrons are channeled to HdrD for reduction of the second heterodisulfide.