Formate-Dependent Heterodisulfide Reduction in aMethanomicrobialesArchaeon

Abstract

Hydrogenotrophic methanogens produce CH4 using H2 as an electron donor to reduce CO2. In the absence of H2, many are able to use formate or alcohols as alternate electron donors. Methanogens from the order Methanomicrobiales are capable of growth with H2, but many lack genes encoding hydrogenases that are typically found in other hydrogenotrophic methanogens. In an effort to better understand electron flow in methanogens from the Methanomicrobiales, we undertook a genetic and biochemical study of heterodisulfide reductase (Hdr) in Methanoculleus thermophilus. Hdr catalyzes an essential reaction by coupling the first and last steps of methanogenesis through flavin-based electron bifurcation. Hdr from M. thermophilus co-purified with formate dehydrogenase (Fdh) and only displayed activity when formate was supplied as an electron donor. We found no evidence of an Hdr associated hydrogenase, and H2 could not function as an electron donor, even with Hdr purified from cells grown on H2. We found that cells catalyze a formate hydrogenlyase activity that is likely essential for generating the formate needed for the Hdr reaction. Together, these results highlight the importance of formate as an electron donor for methanogenesis and suggest the ability to use formate is closely integrated into the methanogenic pathway in organisms from the order Methanomicrobiales. Importance Methanogens from the order Methanomicrobiales are thought to prefer H2 as an electron donor for growth. They are ubiquitous in anaerobic environments such as in wastewater treatment facilities, anaerobic digesters, and the rumen where they catalyze the terminal steps in the breakdown of organic matter. However, despite their importance, the metabolism of these organisms remains understudied. Using a genetic and biochemical approach, we show that formate metabolism is closely integrated into methanogenesis in Methanoculleus thermophilus. This is due to a requirement for formate as the electron donor to heterodisulfide reductase (Hdr), an enzyme responsible for catalyzing essential reactions in methanogenesis by linking the initial CO2 fixing step to the exergonic, terminal reaction of the pathway. These results suggest that hydrogen is not necessarily the preferred electron donor for all hydrogenotrophic methanogens and provide insight into the metabolism of methanogens from the order Methanomicrobiales.

Three-megadalton complex of methanogenic electron-bifurcating and CO 2 -fixing enzymes

“…These reactions are catalyzed by the molybdenum-containing formyl-methanofuran dehydrogenase, FmdABCDFG, or its tungsten isoform, FwdABCDFG ( 3 , 4 ). Electrons for CO 2 reduction are provided by methyl viologen–reducing hydrogenase (MvhAG) ( 5 – 7 ) or formate dehydrogenase (FdhAB) ( 8 – 11 ) in complex with heterodisulfide reductase (HdrABC), which carries out flavin-based electron bifurcation (FBEB) ( 5 ). In this process, the energy in a pair of electrons is split, giving rise to a strongly reducing electron for CO 2 fixation and a weakly reducing electron for reduction of the heterodisulfide of coenzyme M and coenzyme B (CoM-S-S-CoB), a co-oxidant that is regenerated by the methane formation reaction [for reviews, see ( 12 , 13 )].…”

“…A generally accepted scheme of hydrogenotrophic methanogenesis features Mvh-Hdr or Fdh-Hdr, and Fmd or Fwd as independent complexes, which are linked by electron transfer by a diffusible ferredoxin ( 1 , 11 , 14 ). However, members of the class Methanomicrobia, including Methanospirillum hungatei , encode all or some Fmd genes in the Hdr gene cluster (fig.…”

“…predicted from the genetic context that Mvh, Hdr, and Fwd in Methanocella conradii form a large enzyme complex ( 15 ). This hypothesis was partially supported by pull-down assays that indicated the presence of transient complexes of Fdh-Hdr-Fwd and Mvh-Hdr-Fwd in Methanococcus maripaludis and Fdh-Hdr-Fwd in Methanoculleus thermophilus ( 9 – 11 ), although these complexes were not stable to size exclusion chromatography (SEC) and the whole complexes were not enzymatically characterized.…”

Process of energy conservation in the extremely haloalkaliphilic methyl‐reducing methanogen Methanonatronarchaeum thermophilum

“…In some hydrogenotrophic methanogenic archaea, cytoplasmic forms of formate dehydrogenase were described, which transfer electrons to F 420 [39–42]. Other soluble formate dehydrogenases are coupled to the HdrABC complex and are involved in Fd red formation [43–45]. Hence, cytoplasmic formate dehydrogenases play an important role in obligate hydrogenotrophic methanogens (reduction of F 420 , Fd ox , or CoM‐S‐S‐CoB [13]), but none of these cytoplasmic enzymes is involved in respiratory electron transport.…”

Complete Genome Sequence of the Secondary Alcohol-Utilizing Methanogen Methanospirillum hungatei Strain GP1

“…There is a single putative coenzyme F 420 -dependent secondary alcohol dehydrogenase that shares 79% amino acid identity with the alcohol dehydrogenase of Methanoculleus thermophilus strain TCI ( 4 ). Recent data suggest that methanogens from the order Methanomicrobiales can oxidize F 420 H 2 and produce formate through the action of a reversible F 420 -dependent formate dehydrogenase ( 12 ). It may be that alcohol oxidation ultimately leads to formate production before CO 2 reduction occurs.…”