A periplasmic flavoprotein in Wolinella succinogenes that resembles the fumarate reductase of Shewanella putrefaciens

Simon, Jörg; Groß, Roland; Klimmek, Oliver; Ringel, Michael; Kröger, Achim

doi:10.1007/s002030050593

Cited by 29 publications

(17 citation statements)

References 52 publications

(57 reference statements)

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“…The nrfA gene encodes the precursor of the catalytic subunit of nitrite reductase (507 residues), which includes a signal peptide of 22 residues; this suggests translocation of the protein into the periplasm by the Sec system. Residues 23–41 predicted by nrfA are identical to the N‐terminal 19 residues of the mature catalytic subunit (Simon et al ., 1998a). NrfA is predicted to be a hydrophilic protein and to carry five haem C groups, four of which are bound by the cysteine residues of the common CxxCH motif.…”

Section: Resultssupporting

confidence: 82%

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

Simon

Groß

Einsle

et al. 2000

Molecular Microbiology

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145

190

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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.

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Section: Resultssupporting

confidence: 82%

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

Simon

Groß

Einsle

et al. 2000

Molecular Microbiology

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145

190

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“…This is consistent with the fact that in all other organisms in which fumarate reduction has been studied, the fumarate reductase is either cytoplasmic or periplasmic (7,8,32,41,49). Thus, outer membrane cytochromes should not be involved in fumarate respiration.…”

Section: Physiological Role Of Omcb and Omcc And Implications Forsupporting

confidence: 77%

OmcB, ac-Type Polyheme Cytochrome, Involved in Fe(III) Reduction inGeobacter sulfurreducens

2003

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Microorganisms in the family Geobacteraceae are the predominant Fe(III)-reducing microorganisms in a variety of subsurface environments in which Fe(III) reduction is an important process, but little is known about the mechanisms for electron transport to Fe(III) in these organisms. The Geobacter sulfurreducens genome was found to contain a 10-kb chromosomal duplication consisting of two tandem three-gene clusters. The last genes of the two clusters, designated omcB and omcC, encode putative outer membrane polyheme c-type cytochromes which are 79% identical. The role of the omcB and omcC genes in Fe(III) reduction in G. sulfurreducens was investigated. OmcB and OmcC were both expressed during growth with acetate as the electron donor and either fumarate or Fe(III) as the electron acceptor. OmcB was ca. twofold more abundant under both conditions. Disrupting omcB or omcC by gene replacement had no impact on growth with fumarate. However, the OmcB-deficient mutant was greatly impaired in its ability to reduce Fe(III) both in cell suspensions and under growth conditions. In contrast, the ability of the OmcC-deficient mutant to reduce Fe(III) was similar to that of the wild type. When omcB was reintroduced into the OmcB-deficient mutant, the capacity for Fe(III) reduction was restored in proportion to the level of OmcB production. These results indicate that OmcB, but not OmcC, has a major role in electron transport to Fe(III) and suggest that electron transport to the outer membrane is an important feature in Fe(III) reduction in this organism.Microbial oxidation of organic matter coupled to the reduction of Fe(III) plays an important role in the degradation of natural and contaminant organic matter in aquatic sediments and the subsurface, and this process influences the mobility of a variety of trace metals in these environments (for reviews, see references 23 and 24). Insoluble Fe(III) oxides, which are the primary form of Fe(III) in most soils and sediments, are unlike commonly considered electron acceptors such as oxygen, nitrate, fumarate, sulfate, and carbon dioxide, which are soluble and can readily enter a cell to be reduced. If, as is generally considered to be the case (23, 43), Fe(III)-reducing microorganisms reduce extracellular Fe(III), then electron transport to Fe(III) is likely to proceed via mechanisms significantly different from those for the reduction of soluble electron acceptors.Furthermore, phylogenetically distinct Fe(III) reducers might reduce Fe(III) oxides via significantly different mechanisms. For example, Geothrix fermentans and Shewanella species produce soluble electron shuttles and/or Fe(III)-chelating compounds which alleviate the need for contact between cells and insoluble Fe(III) for Fe(III) reduction (38)(39)(40)54). Others, such as Geobacter metallireducens, do not produce electron shuttles or Fe(III) chelators (37) but utilize appendages such as flagella and pili to search for or to make direct contact with insoluble Fe(III) (6).The mechanisms for Fe(III) reduction in Geobacte...

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“…The NrfHA complex was isolated from the membrane fraction of W. succinogenes . The preparation consists of two polypeptides previously identified by N‐terminal sequencing as the nrfH and nrfA gene products [4,16]. Both proteins are c ‐type cytochromes as revealed by heme staining of an SDS/polyacrylamide gel [4].…”

Section: Resultsmentioning

confidence: 99%

The tetraheme cytochrome c NrfH is required to anchor the cytochrome c nitrite reductase (NrfA) in the membrane of Wolinella succinogenes

Simon

Pisa

Stein

et al. 2001

European Journal of Biochemistry

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The electron-transport chain that catalyzes nitrite respiration with formate in Wolinella succinogenes consists of formate dehydrogenase, menaquinone and the nitrite reductase complex. The latter catalyzes nitrite reduction by menaquinol and is made up of NrfA and NrfH, two c-type cytochromes. NrfA is the catalytic subunit; its crystal structure is known. NrfH belongs to the NapC/NirT family of membrane-bound c-type cytochromes and mediates electron transport between menaquinol and NrfA. It is demonstrated here by MALDI MS that four heme groups are attached to NrfH. A DnrfH deletion mutant of W. succinogenes was constructed by replacing the nrfH gene with a kanamycin-resistance gene cartridge. This mutant did not form the NrfA protein, probably because of a polar effect of the mutation on nrfA expression. The nrfHAIJ gene cluster was restored by integration of an nrfH-containing plasmid into the genome of the DnrfH mutant. The resulting strain had wild-type properties with respect to growth by nitrite respiration and nitrite reductase activity. A mutant (stopH) that contained the nrfHAIJ locus with nrfH modified by two artificial stop codons near its 5 0 end produced wild-type amounts of NrfA in the absence of the NrfH protein. NrfA was located exclusively in the soluble cell fraction of the stopH mutant, indicating that NrfH acts as the membrane anchor of the NrfHA complex in wild-type bacteria. The stopH mutant did not grow by nitrite respiration and did not catalyze nitrite reduction by formate, indicating that the electron transport is strictly dependent on NrfH. The NrfH protein seems to be an unusual member of the NapC/NirT family as it forms a stable complex with its redox partner protein NrfA.Keywords: cytochrome c nitrite reductase; NapC/NirT family; nitrite respiration; Wolinella succinogenes.The epsilon proteobacterium Wolinella succinogenes can grow by anaerobic respiration using formate as electron donor and nitrite as electron acceptor [1]. The electron transport chain that catalyzes nitrite reduction by formate is integrated into the bacterial membrane and consists of formate dehydrogenase, menaquinone and the heterodimeric cytochrome c nitrite reductase complex (NrfHA) [2][3][4]. The purified complex of NrfH and NrfA catalyzed nitrite reduction to ammonia by the soluble menaquinol analog 2,3-dimethyl-1,4-naphthoquinol, whereas, on its own, the catalytic subunit NrfA reacted only with artificial electron donors such as reduced viologen dyes [4,5]. Proteoliposomes containing the NrfHA complex together with menaquinone and formate dehydrogenase catalyzed nitrite reduction by formate which was coupled to the generation of an electrochemical proton potential across the liposomal membrane [4]. The crystal structure of NrfA, a pentaheme cytochrome c, was recently determined [6].NrfH apparently mediates the electron transfer from menaquinol located in the membrane to NrfA. The NrfH protein is a c-type cytochrome with four CXXCH heme c binding motifs, which is encoded by the first gene of the nrfHAIJ locu...

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A periplasmic flavoprotein in Wolinella succinogenes that resembles the fumarate reductase of Shewanella putrefaciens

Cited by 29 publications

References 52 publications

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

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

OmcB, ac-Type Polyheme Cytochrome, Involved in Fe(III) Reduction inGeobacter sulfurreducens

The tetraheme cytochrome c NrfH is required to anchor the cytochrome c nitrite reductase (NrfA) in the membrane of Wolinella succinogenes

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