Physiological role of the F 420 -non-reducing hydrogenase (Mvh) from Methanothermobacter marburgensis

Abstract

F(420)-non-reducing hydrogenase (Mvh) from Methanothermobacter marburgensis is a [NiFe] hydrogenase composed of the three subunits MvhA, MvhG, and MvhD. Subunits MvhA and MvhG form the basic hydrogenase module conserved in all [NiFe] hydrogenases, whereas the 17-kDa MvhD subunit is unique to Mvh. The function of this extra subunit is completely unknown. In this work, the physiological function of this hydrogenase, and in particular the role of the MvhD subunit, is addressed. In cells of Mt. marburgensis from Ni(2+)-limited chemostat cultures the amount of Mvh decreased about 70-fold. However, the amounts of mvh transcripts did not decrease in these cells as shown by competitive RT-PCR, arguing against a regulation at the level of transcription. In cells grown in the presence of non-limiting amounts of Ni(2+), Mvh was found in two chromatographically distinct forms-a free form and in a complex with heterodisulfide reductase. In cells from Ni(2+)-limited chemostat cultures, Mvh was only found in a complex with heterodisulfide reductase. The EPR spectrum of the purified enzyme reduced with sodium dithionite was dominated by a signal with g(zyx)=2.006, 1.936 and 1.912. The signal could be observed at temperatures up to 80 K without broadening, indicative of a [2Fe-2S] cluster. Subunit MvhD contains five cysteine residues that are conserved in MvhD homologues of other organisms. Four of these conserved cysteine residues can be assumed to coordinate the [2Fe-2S] cluster that was detected by EPR spectroscopy. The MvhG subunit contains 12 cysteine residues, which are known to ligate three [4Fe-4S] clusters. Data base searches revealed that in some organisms, including the Methanosarcina species and Archaeoglobus fulgidus, a homologue of mvhD is fused to the 3' end of an hdrA homologue, which encodes a subunit of heterodisulfide reductase. These data allow the conclusion that the only function of Mvh is to provide reducing equivalents for heterodisulfide reductase and that the MvhD subunit is an electron transfer protein that forms the contact site to heterodisulfide reductase.

Protein complexing in a methanogen suggests electron bifurcation and electron delivery from formate to heterodisulfide reductase

“…The coelution of these proteins with three separate protein subunits under three different growth conditions strongly suggests that the observed complex exists in vivo and may be involved in energy conservation. The interaction of Hdr with F 420 -nonreducing hydrogenase was suggested previously in studies of these enzymes in Methanothermobacter marburgensis (15,16). Indeed, in some methanogens the α subunit of Hdr is fused to the δ subunit of the F 420 -nonreducing hydrogenase (16).…”

“…The interaction of Hdr with F 420 -nonreducing hydrogenase was suggested previously in studies of these enzymes in Methanothermobacter marburgensis (15,16). Indeed, in some methanogens the α subunit of Hdr is fused to the δ subunit of the F 420 -nonreducing hydrogenase (16). However, the presence of Fdh and Fwd in the complex with Hdr is unique.…”

“…In fact, an interaction between Hdr and Fwd was first proposed in 1988 (22) and was later implicated in potential flavin-based electron bifurcation by Hdr to drive the first step in methanogenesis (1). Because previous Hdr purifications did not show any interaction between Hdr and Fwd (15,16), it is likely that the physical interaction between these two proteins is weak, and our method of purification, which involved minimal manipulation, was able to retain Fwd bound to Hdr. HdrA is known to contain one mole of FAD per mole of HdrA (15), and it is likely that, through flavin-based electron bifurcation, HdrA can reduce ferredoxin that is then used by Fwd.…”

“…Previously, only the F 420 -nonreducing hydrogenase was thought to deliver electrons to Hdr. Hdr purified from M. marburgensis was observed to associate with an F 420 -nonreducing hydrogenase, but not with Fdh (15,16). However, this organism is not known to grow with formate as the sole electron donor (17).…”

Genome sequencing of a single cell of the widely distributed marine subsurface Dehalococcoidia, phylum Chloroflexi

“…This cluster was one of the longest and most obvious stretches of ORFs identified (encoding at least 20 ORFs) in our data set, whereby no homologues in known DEH could be identified by BLASTP analyses, yet was downstream of known DEH genes. We hypothesise that the heterodisulfide reductase-like enzymes have important roles in cytoplasmic electron transfer and energy conserving mechanisms, like in other anaerobes such as sulphate reducers, acetogens and methanogens (Stojanowic et al, 2003;Strittmatter et al, 2009;Kaster et al, 2011b;Callaghan et al, 2012). These complexes might be especially important for transferring reducing equivalents released during beta-oxidation and/or conversions of succinate to acetyl-CoA (via the methylmalonyl-CoA pathway), to and from ferredoxins or NADH, possibly by electron bifurcating/confurcating mechanisms that may be linked to other metabolic steps (Buckel and Thauer, 2012;Grein et al, 2012).…”