A NapC/NirT‐type cytochrome c (NrfH) is the mediator between the quinone pool and the cytochrome c nitrite reductase of Wolinella succinogenes

Abstract

Wolinella succinogenes can grow by anaerobic respiration with nitrate or nitrite using formate as electron donor. Two forms of nitrite reductase were isolated from the membrane fraction of W. succinogenes. One form consisted of a 58 kDa polypeptide (NrfA) that was identical to the periplasmic nitrite reductase. The other form consisted of NrfA and a 22 kDa polypeptide (NrfH). Both forms catalysed nitrite reduction by reduced benzyl viologen, but only the dimeric form catalysed nitrite reduction by dimethylnaphthoquinol. Liposomes containing heterodimeric nitrite reductase, formate dehydrogenase and menaquinone catalysed the electron transport from formate to nitrite; this was coupled to the generation of an electrochemical proton potential (positive outside) across the liposomal membrane. It is concluded that the electron transfer from menaquinol to the catalytic subunit (NrfA) of W. succinogenes nitrite reductase is mediated by NrfH. The structural genes nrfA and nrfH were identified in an apparent operon (nrfHAIJ) with two additional genes. The gene nrfA encodes the precursor of NrfA carrying an N‐terminal signal peptide (22 residues). NrfA (485 residues) is predicted to be a hydrophilic protein that is similar to the NrfA proteins of Sulfurospirillum deleyianum and of Escherichia coli. NrfH (177 residues) is predicted to be a membrane‐bound tetrahaem cytochrome c belonging to the NapC/NirT family. The products of nrfI and nrfJ resemble proteins involved in cytochrome c biogenesis. The C‐terminal third of NrfI (902 amino acid residues) is similar to CcsA proteins from Gram‐positive bacteria, cyanobacteria and chloroplasts. The residual N‐terminal part of NrfI resembles Ccs1 proteins. The deduced NrfJ protein resembles the thioredoxin‐like proteins (ResA) of Helicobacter pylori and of Bacillus subtilis, but lacks the common motif CxxC of ResA. The properties of three deletion mutants of W. succinogenes (ΔnrfJ,ΔnrfIJ and ΔnrfAIJ) were studied. Mutants ΔnrfAIJ and ΔnrfIJ did not grow with nitrite as terminal electron acceptor or with nitrate in the absence of NH4+ and lacked nitrite reductase activity, whereas mutant ΔnrfJ showed wild‐type properties. The NrfA protein formed by mutant ΔnrfIJ seemed to lack part of the haem C, suggesting that NrfI is involved in NrfA maturation.

Characterization of the Menaquinone Reduction Site in the Diheme Cytochrome b Membrane Anchor of Wolinella succinogenes NiFe-hydrogenase

“…Modified pHydcat plasmids were sequenced to confirm the mutations. Nitrate-grown cells of W. succinogenes ⌬hydABC were used for transformation as described previously (13,26). Transformants were selected on agar plates containing a medium with formate and nitrate as energy substrates as well as kanamycin (25 mg/liter) and chloramphenicol (12.5 mg/liter).…”

“…The ⑀-proteobacterium Wolinella succinogenes grows by anaerobic respiration using hydrogen or formate as electron donor and various organic and inorganic compounds as electron acceptor such as fumarate, nitrate, nitrite, or polysulfide (11)(12)(13)(14)(15). The NiFe-hydrogenase of W. succinogenes is a membranebound heterotrimeric complex oriented to the periplasmic space that consists of two hydrophilic (HydA and HydB) and one hydrophobic subunit (HydC).…”

C-type cytochromes: diverse structures and biogenesis systems pose evolutionary problems

“…(Note that in some bacteria, the analogues of these two parts of E. coli CcmH are transcribed from separate genes (Thö ny-Meyer 1997).) Interestingly, in W. succinogenes, which uses the system II cytochrome c biogenesis apparatus, it is proposed that haem attachment to NrfA requires the additional dedicated system II gene nrfI; this gene has no obvious homology to nrfEFG (Simon et al 2000;Kranz et al 2002;Pisa et al 2002).…”

Genomic and in situ investigations of the novel uncultured Chloroflexi associated with 0092 morphotype filamentous bulking in activated sludge

“…Potential contribution of the B45 phylotype to denitrification No putative nitrate reductase gene was annotated in the Cfx-K genome, although the presence of a putative cytochrome c nitrite reductase (NrfA) suggests an ability for dissimilatory nitrite reduction to ammonia (Simon et al, 2000). No nitric oxide reductase (NorBC) encoding gene was detected either, although nitrite reductase (NrfA) has been shown to reduce nitric oxide to ammonia, a reaction that was suggested to be associated with protection from nitrosative stress (Poock et al, 2002).…”