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

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 Delta nrfH 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 Delta nrfH 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' 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.

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

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 (DeltanrfJ, DeltanrfIJ and DeltanrfAIJ) were studied. Mutants DeltanrfAIJ and DeltanrfIJ did not grow with nitrite as terminal electron acceptor or with nitrate in the absence of NH4+ and lacked nitrite reductase activity, whereas mutant DeltanrfJ showed wild-type properties. The NrfA protein formed by mutant DeltanrfIJ seemed to lack part of the haem C, suggesting that NrfI is involved in NrfA maturation.

HNCAN pulse sequences for sequential backbone resonance assignment across proline residues in perdeuterated proteins.

A TROSY-based triple-resonance pulse scheme is described which correlates backbone 1H and 15N chemical shifts of an amino acid residue with the 15N chemical shifts of both the sequentially preceding and following residues. The sequence employs 1J(NC alpha) and 2J(NC alpha) couplings in two sequential magnetization transfer steps in an 'out-and-back' manner. As a result, N,N connectivities are obtained irrespective of whether the neighbouring amide nitrogens are protonated or not, which makes the experiment suitable for the assignment of proline resonances. Two different three-dimensional variants of the pulse sequence are presented which differ in sensitivity and resolution to be achieved in one of the nitrogen dimensions. The new method is demonstrated with two uniformly 2H/13C/15N-labelled proteins in the 30-kDa range.

The single cysteine residue of the Sud protein is required for its function as a polysulfide-sulfur transferase in Wolinella succinogenes.

The periplasmic Sud protein which is induced in Wolinella succinogenes growing by polysulfide respiration, has been previously proposed to serve as a polysulfide binding protein and to transfer polysulfide-sulfur to the active site of polysulfide reductase [Klimmek, O, Kreis, V., Klein, C., Simon, J., Wittershagen, A. & Kröger, A. (1998) Eur. J. Biochem. 253, 263-269.]. The results presented in this communication suggest that polysulfide-sulfur is covalently bound to the single cysteine residue (Cys109) of the Sud monomer, and that Cys109 is required for tight binding of polysulfide-sulfur and for sulfur transfer. A modified Sud protein [(C109S)Sud-His6] in which the cysteine residue was replaced by serine, did not catalyze sulfur transfer from polysulfide to cyanide and did not stimulate electron transport to polysulfide, in contrast to Sud-His6. The polysulfide-sulfur bound to (C109S)Sud-His6 was fully removed upon dialysis against sulfide. After this treatment, Sud-His6 retained one sulfur atom per monomer; thiocyanate was formed upon addition of cyanide to the preparation. After incubation of Sud-His6 with polysulfide, a proportion of the Sud-His6 monomers carried one or two sulfur atoms, as shown by matrix-assisted laser desorption ionization mass spectrometry. The sulfur atoms were absent from monomers derived from Sud-His6 treated with cyanide and from (C109S)Sud-His6 incubated with polysulfide.

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

During growth with fumarate as the terminal electron transport acceptor and either formate or sulfide as the electron donor, Wolinella succinogenes induced a peri-plasmic protein (54 kDa) that reacted with an antiserum raised against the periplasmic fumarate reductase (Fcc) of Shewanella putrefaciens. However, the periplasmic cell fraction of W. succinogenes did not catalyze fumarate reduction with viologen radicals. W. succinogenes grown with polysulfide instead of fumarate contained much less (< 10%) of the 54-kDa antigen, and the antigen was not detectable in nitrate-grown bacteria. The antigen was most likely encoded by the fccA gene of W. succinogenes. The antigen was absent from a DeltafccABC mutant, and its size is close to that of the protein predicted by fccA. The fccA gene probably encodes a pre-protein carrying an N-terminal signal peptide. The sequence of the mature FccA (481 residues, 52.4 kDa) is similar (31% identity) to that of the C-terminal part (450 residues) of S. putrefaciens fumarate reductase. As indicated by Northern blot analysis, fccA is cotranscribed with fccB and fccC. The proteins predicted from the fccB and fccC gene sequences represent tetraheme cytochromes c. FccB is similar to the N-terminal part (150 residues) of S. putrefaciens fumarate reductase, while FccC resembles the tetraheme cytochromes c of the NirT/NapC family. The DeltafccABC mutant of W. succinogenes grew with fumarate and formate or sulfide, suggesting that the deleted proteins were not required for fumarate respiration with either electron donor.

The function of the periplasmic Sud protein in polysulfide respiration of Wolinella succinogenes.

The periplasmic Sud protein was previously isolated as a sulfide dehydrogenase from Wolinella succinogenes. Sud modified by a C-terminal His-tag (Sud-His6) was produced in Escherichia coli by expression of the sud gene. Sud-His6 catalyzed thiocyanate formation from cyanide and polysulfide. The Vmax of this activity was more than one order of magnitude higher than that of sulfide oxidation by dimethyl-naphthoquinone and that of polysulfide reduction by BH4-. The apparent Km was less than 20 microM polysulfide. Polysulfide and not elemental sulfur was found to be the product of sulfide oxidation by dimethyl-naphthoquinone, in contrast to the earlier view [Kreis-Kleinschmidt, V., Fahrenholz, F., Kojro, E. & Kröger. A. (1995) Arch. Microbiol. 165, 65-68]. Sud-His6 did not contain metal ions or other prosthetic groups. Replacement by site-directed mutagenesis of the single cysteine residue of the Sud monomer caused complete loss of activity, while the exchange of the single histidine residue or of the lysine residue situated next to cysteine did not affect activity. In equilibrium dialysis, the Sud-His6 monomer bound up to ten polysulfide sulfur atoms with a dissociation constant of 0.2 mM. Sud-His6 loaded with polysulfide sulfur showed an absorption spectrum in the range of 350-400 nm; this spectrum differed from that of free polysulfide. Electron transport from H2 to polysulfide catalyzed by the membrane fraction of W. succinogenes was stimulated by the presence of small amounts of Sud-His6. The apparent Km for polysulfide decreased sevenfold in the presence of saturating amounts of Sud-His6 (1 microM Sud-His6 dimer). Similar results were obtained with intact W. succinogenes cells containing low and high amounts of Sud. Sud appears to function as a polysulfide binding protein and probably binds polysulfide sulfur to its cysteine residue and transfers it to the substrate site of the membraneous polysulfide reductase.

Flavodoxin from Wolinella succinogenes.

A monomeric flavoprotein (18.8 kDa) was isolated from the soluble cell fraction of Wolinella succinogenes and was identified as a flavodoxin based on its N-terminal sequence, FMN content, and redox properties. The midpoint potentials of the flavodoxin (Fld) at pH 7. 5 were measured as -95 mV (Fldox/Flds) and -450 mV (Flds/Fldred) relative to the standard hydrogen electrode. The cellular flavodoxin content [0.3 micromol (g protein)-1] was the same in bacteria grown with fumarate or with polysulfide as the terminal acceptor of electron transport. The flavodoxin did not accept electrons from hydrogenase or formate dehydrogenase, the donor enzymes of electron transport to fumarate or polysulfide. Pyruvate:flavodoxin oxidoreductase activity [180 U (g cellular protein)-1] was detected in the soluble cell fraction of W. succinogenes grown with fumarate or polysulfide. The enzyme was equally active with Fldox or Flds at high concentrations. The Km for Flds (80 microM) was larger than that for Fldox and for the ferredoxin isolated from W. succinogenes (15 microM). We conclude that flavodoxin serves anabolic rather than catabolic functions in W. succinogenes.

Properties of a Wolinella succinogenes mutant lacking periplasmic sulfide dehydrogenase (Sud).

A delta sud deletion mutant of Wolinella succinogenes that lacked the periplasmic sulfide dehydrogenase (Sud) was constructed using homologous recombination. The mutant grew with sulfide and fumarate, indicating that Sud was not a component of the electron transport chain that catalyzed fumarate respiration with sulfide as an electron donor. Likewise, growth with formate and either polysulfide or sulfur was not affected by the deletion. Removal of Sud from wild-type W. succinogenes by spheroplast formation did not decrease the activity of electron transport to polysulfide. The delta psr deletion mutant that lacks polysulfide reductase (Psr) grew by fumarate respiration with sulfide as an electron donor, indicating that Psr is not required for this activity.

Cloning and nucleotide sequence of the psrA gene of Wolinella succinogenes polysulphide reductase.

The polysulphide reductase (formerly sulphur reductase) of Wolinella succinogenes is a component of the phosphorylative electron transport system with polysulphide as the terminal acceptor. Using an antiserum raised against the major subunit (PsrA, 85 kDa) of the enzyme, the corresponding gene (psrA) was cloned from a lambda-gene bank. The N-terminal amino acid sequence of PsrA mapped within the psrA gene product, which also contained an apparent signal peptide. Downstream of the psrA gene two more open reading frames (psrB and psrC) were found. The three genes may form a transcriptional unit with the transcription start site in front of psrA. The three genes were present only once on the genome. PsrA is a hydrophilic protein homologous to the largest subunits of six prokaryotic molybdoenzymes. PsrB is predicted to be hydrophilic, to contain ferredoxin-like cysteine clusters and to be homologous to the smaller hydrophilic subunits of four molybdoenzymes. PsrC is predicted to be a hydrophobic protein that could possibly serve as the membrane anchor of the enzyme.