Involvement in denitrification of the napKEFDABC genes encoding the periplasmic nitrate reductase system in the denitrifying phototrophic bacterium Rhodobacter sphaeroides f. sp. denitrificans

Seven genes, napKEFDABC, encoding the periplasmic nitrate reductase system were cloned from the denitrifying phototrophic bacterium Rhodobacter sphaeroides f. sp. denitrificans IL106. Two transmembrane proteins, NapK and NapE, an iron-sulfur protein NapF, a soluble protein NapD, a catalytic subunit of nitrate reductase precursor NapA, a soluble c-type diheme cytochrome precursor NapB, and a membrane-anchored c-type tetraheme cytochrome NapC were deduced as the gene products. Every mutant in which each nap gene was disrupted by omega-cassette insertion lost nitrate reductase activity as well as the ability of cells to grow with nitrate under anaerobic-dark conditions. A transconjugant of the napD-disrupted mutant with a plasmid bearing the napKEFDABC genes recovered both nitrate reductase activity and nitrate-dependent anaerobic-dark growth of cells. Denitrification activity, which was not observed in the napD mutant, was also restored by the conjugation. These results indicate that the periplasmic nitrate reductase encoded by the napKEFDABC genes is the enzyme responsible for denitrification in this phototroph, although the presence of a membrane-bound nitrate reductase has been reported in the same strain.     

Identification of a small tetraheme cytochrome c and a flavocytochrome c as two of the principal soluble cytochromes c in Shewanella oneidensis strain MR1.

Two abundant, low-redox-potential cytochromes c were purified from the facultative anaerobe Shewanella oneidensis strain MR1 grown anaerobically with fumarate. The small cytochrome was completely sequenced, and the genes coding for both proteins were cloned and sequenced. The small cytochrome c contains 91 residues and four heme binding sites. It is most similar to the cytochromes c from Shewanella frigidimarina (formerly Shewanella putrefaciens) NCIMB400 and the unclassified bacterial strain H1R (64 and 55% identity, respectively). The amount of the small tetraheme cytochrome is regulated by anaerobiosis, but not by fumarate. The larger of the two low-potential cytochromes contains tetraheme and flavin domains and is regulated by anaerobiosis and by fumarate and thus most nearly corresponds to the flavocytochrome c-fumarate reductase previously characterized from S. frigidimarina to which it is 59% identical. However, the genetic context of the cytochrome genes is not the same for the two Shewanella species, and they are not located in multicistronic operons. The small cytochrome c and the cytochrome domain of the flavocytochrome c are also homologous, showing 34% identity. Structural comparison shows that the Shewanella tetraheme cytochromes are not related to the Desulfovibrio cytochromes c(3) but define a new folding motif for small multiheme cytochromes c.     

The 1.25 A resolution structure of the diheme NapB subunit of soluble nitrate reductase reveals a novel cytochrome c fold with a stacked heme arrangement

The diheme cytochrome NapB constitutes the small subunit of a periplasmic nitrate reductase found in a wide variety of bacterial species, including pathogens. The NapB protein is essential in transferring electrons to the large catalytic subunit NapA, which subsequently reduces nitrate to nitrite. Here we present the crystal structure of a proteolyzed form of recombinant NapB from Haemophilus influenzae, which was determined by the multiple-wavelength anomalous dispersion (MAD) method at 1.25 A resolution. This structure shows an unprecedented fold, confirming that NapB proteins belong to a new class of cytochromes. The two heme groups have nearly parallel heme planes and are stacked at van der Waals distances with an iron-to-iron distance of only 9.9 A, two structural features that are also present in the split-Soret diheme cytochrome c from Desulfovibrio desulfuricans ATCC 27774, which is otherwise unrelated in the peptide chain folding pattern. The two propionate side chains on both heme groups are hydrogen-bonded to each other, a structural characteristic that to date also has not been reported in any other heme protein. The propionates of one of the heme groups are pulled toward the interior of the molecule due to a salt bridge and a number of hydrogen bonds between the propionates and conserved residues. We propose a hypothetical but plausible model of the NapAB complex in which the four redox centers are positioned in a virtually linear configuration which spans a distance of nearly 40 A, suggesting an efficient pathway for the transfer of electrons from NapC, the physiological electron donor of NapB, to a nitrate molecule at the catalytic site of NapA.     

Variants of the tetrahaem cytochrome c quinol dehydrogenase NrfH characterize the menaquinol-binding site, the haem c-binding motifs and the transmembrane segment

Members of the NapC/NrfH family are multihaem c-type cytochromes that exchange electrons with oxidoreductases situated at the outside of the cytoplasmic membrane or in the periplasmic space of many proteobacteria. They form a group of membrane-bound quinol dehydrogenases that are essential components of several electron transport chains, for example those of periplasmic nitrate respiration and respiratory nitrite ammonification. Knowledge of the structure-function relationships of NapC/NrfH proteins is scarce and only one high-resolution structure (Desulfovibrio vulgaris NrfH) is available. In the present study, several Wolinella succinogenes mutants that produce variants of NrfH, the membrane anchor of the cytochrome c nitrite reductase complex, were constructed and characterized in order to improve the understanding of the putative menaquinol-binding site, the maturation and function of the four covalently bound haem c groups and the importance of the N-terminal transmembrane segment. Based on amino acid sequence alignments, a homology model for W. succinogenes NrfH was constructed that underlines the overall conservation of tertiary structure in spite of a low sequence homology. The results support the proposed architecture of the menaquinol-binding site in D. vulgaris NrfH, demonstrate that each histidine residue arranged in one of the four CX(2)CH haem c-binding motifs is essential for NrfH maturation in W. succinogenes, and indicate a limited flexibility towards the length and structure of the transmembrane region.     

Electron transport to periplasmic nitrate reductase (NapA) of Wolinella succinogenes is independent of a NapC protein

The rumen bacterium Wolinella succinogenes grows by respiratory nitrate ammonification with formate as electron donor. Whereas the enzymology and coupling mechanism of nitrite respiration is well known, nitrate reduction to nitrite has not yet been examined. We report here that intact cells and cell fractions catalyse nitrate and chlorate reduction by reduced viologen dyes with high specific activities. A gene cluster encoding components of a putative periplasmic nitrate reductase system (napA, G, H, B, F, L, D) was sequenced. The napA gene was inactivated by inserting a kanamycin resistance gene cassette. The resulting mutant did not grow by nitrate respiration and did not reduce nitrate during growth by fumarate respiration, in contrast to the wild type. An antigen was detected in wild-type cells using an antiserum raised against the periplasmic nitrate reductase (NapA) from Paracoccus pantotrophus. This antigen was absent in the W. succinogenes napA mutant. It is concluded that the periplasmic nitrate reductase NapA is the only respiratory nitrate reductase in W. succinogenes, although a second nitrate-reducing enzyme is apparently induced in the napA mutant. The nap cluster of W. succinogenes lacks a napC gene whose product is thought to function in quinol oxidation and electron transfer to NapA in other bacteria. The W. succinogenes genome encodes two members of the NapC/NirT family, NrfH and FccC. Characterization of corresponding deletion mutants indicates that neither of these two proteins is required for nitrate respiration. A mutant lacking the genes encoding respiratory nitrite reductase (nrfHA) had wild-type properties with respect to nitrate respiration. A model of the electron transport chain of nitrate respiration is proposed in which one or more of the napF, G, H and L gene products mediate electron transport from menaquinol to the periplasmic NapAB complex. Inspection of the W. succinogenes genome sequence suggests that ammonia formation from nitrate is catalysed exclusively by periplasmic respiratory enzymes.     

Need for cytochrome bc1 complex for dissimilatory nitrite reduction of Pseudomonas aeruginosa

Pseudomonas aeruginosa strains deficient in the genes for cytochrome c1, a subunit of the cytochrome bc1 complex, or the tetraheme membrane protein NapC, which is similar to NirT of Pseudomonas stutzeri, were constructed and their growth was investigated. The cytochrome c1 mutant could not grow under anaerobic conditions with nitrite as an electron acceptor and did not reduce nitrite in spite of its producing active nitrite reductase. NirM (cytochrome c551) and azurin, which are the direct electron donors for nitrite reductase, were reduced by succinate in the presence of the membrane fraction from the wild-type strain as a mediator but not in the presence of that from the cytochrome c1 mutant. These results indicated that cytochrome bc1 complex was necessary for electron transfer from the membrane quinone pool to nitrite reductase. The NapC mutant grew anaerobically at the expense of nitrite, indicating that NapC was not necessary for nitrite reduction.     

Cytochrome components of nitrate- and sulfate-respiring Desulfovibrio desulfuricans ATCC 27774.

Three multiheme c-type cytochromes--the tetraheme cytochrome c3 (molecular weight [MW] 13,500), a dodecaheme cytochrome c (MW 40,800), and a "split-Soret" cytochrome c (MW 51,540), which is a dimer with 2 hemes per subunit (MW 26,300)--were isolated from the soluble fraction of Desulfovibrio desulfuricans (ATCC 27774) grown under nitrate- or sulfate-respiring conditions. Two of them, the dodecaheme and the split-Soret cytochromes, showed no similarities to any of the c-type cytochromes isolated from other sulfate-reducing bacteria, while the tetraheme cytochrome c3 appeared to be analogous to the cytochrome c3 found in other sulfate-reducing bacteria. For all three multiheme c-type cytochromes isolated, the homologous proteins from nitrate- and sulfate-grown cells were indistinguishable in amino acid composition, physical properties, and spectroscopic characteristics. It therefore appears that the same c-type cytochrome components are present when D. desulfuricans ATCC 27774 cells are grown under either condition. This is in contrast to the considerable difference found in Pseudomonas perfectomarina (Liu et al., J. Bacteriol. 154:278-286, 1983), a marine denitrifier, when the cells are grown on nitrate or oxygen as the terminal electron acceptor. In addition, two spectroscopy methods capable of revealing minute structural variations in proteins provided identical information about the tetraheme cytochrome c3 from nitrate-grown and sulfate-grown cells.     

Gene expression analysis of the mechanism of inhibition of Desulfovibrio vulgaris Hildenborough by nitrate-reducing, sulfide-oxidizing bacteria

Sulfate-reducing bacteria (SRB) are inhibited by nitrate-reducing, sulfide-oxidizing bacteria (NR-SOB) in the presence of nitrate. This inhibition has been attributed either to an increase in redox potential or to production of nitrite by the NR-SOB. Nitrite specifically inhibits the final step in the sulfate reduction pathway. When the NR-SOB Thiomicrospira sp. strain CVO was added to mid-log phase cultures of the SRB Desulfovibrio vulgaris Hildenborough in the presence of nitrate, sulfate reduction was inhibited. Strain CVO reduced nitrate and oxidized sulfide, with transient production of nitrite. Sulfate reduction by D. vulgaris resumed once nitrite was depleted. A DNA macroarray with open reading frames encoding enzymes involved in energy metabolism of D. vulgaris was used to study the effects of NR-SOB on gene expression. Shortly following addition of strain CVO, D. vulgaris genes for cytochrome c nitrite reductase and hybrid cluster proteins Hcp1 and Hcp2 were upregulated. Genes for sulfate reduction enzymes, except those for dissimilatory sulfite reductase, were downregulated. Genes for the membrane-bound electron transferring complexes QmoABC and DsrMKJOP were downregulated and unaffected, respectively, whereas direct addition of nitrite downregulated both operons. Overall the gene expression response of D. vulgaris upon exposure to strain CVO and nitrate resembled that observed upon direct addition of nitrite, indicating that inhibition of SRB is primarily due to nitrite production by NR-SOB.     

Roles of NapF, NapG and NapH, subunits of the Escherichia coli periplasmic nitrate reductase, in ubiquinol oxidation

The nap operon of Escherichia coli K-12, encoding a periplasmic nitrate reductase (Nap), encodes seven proteins. The catalytic complex in the periplasm, NapA-NapB, is assumed to receive electrons from the quinol pool via the membrane-bound cytochrome NapC. Like NapA, B and C, a fourth polypeptide, NapD, is also essential for Nap activity. However, none of the remaining three polypeptides, NapF, G and H, which are predicted to encode non-haem, iron-sulphur proteins, are essential for Nap activity, and their function is currently unknown. The relative rates of growth and electron transfer from physiological substrates to Nap have been investigated using strains defective in the two membrane-bound nitrate reductases, and also defective in either ubiquinone or menaquinone biosynthesis. The data reveal that Nap is coupled more effectively to menaquinol oxidation than to ubiquinol oxidation. Conversely, parallel experiments with a second set of mutants revealed that nitrate reductase A couples more effectively with ubiquinol than with menaquinol. Three further sets of strains were constructed with combinations of in frame deletions of ubiCA, menBC, napC, napF and napGH genes. NapF, NapG and NapH were shown to play no role in electron transfer from menaquinol to the NapAB complex but, in the Ubi+Men- background, deletion of napF, napGH or napFGH all resulted in total loss of nitrate-dependent growth. Electron transfer from ubiquinol to NapAB was totally dependent upon NapGH, but not on NapF. NapC was essential for electron transfer from both ubiquinol and menaquinol to NapAB. The results clearly established that NapG and H, but not NapF, are essential for electron transfer from ubiquinol to NapAB. The decreased yield of biomass resulting from loss of NapF in a Ubi+Men+ strain implicates NapF in an energy- conserving role coupled to the oxidation of ubiquinol. We propose that NapG and H form an energy- conserving quinol dehydrogenase functioning as either components of a proton pump or in a Q cycle, as electrons are transferred from ubiquinol to NapC.     

Identification of 42 possible cytochrome C genes in the Shewanella oneidensis genome and characterization of six soluble cytochromes

Through pattern matching of the cytochrome c heme-binding site (CXXCH) against the genome sequence of Shewanella oneidensis MR-1, we identified 42 possible cytochrome c genes (27 of which should be soluble) out of a total of 4758. However, we found only six soluble cytochromes c in extracts of S. oneidensis grown under several different conditions: (1) a small tetraheme cytochrome c, (2) a tetraheme flavocytochrome c-fumarate reductase, (3) a diheme cytochrome c4, (4) a monoheme cytochrome c5, (5) a monoheme cytochrome c', and (6) a diheme bacterial cytochrome c peroxidase. These cytochromes were identified either through N-terminal or complete amino acid sequence determination combined with mass spectroscopy. All six cytochromes were about 10-fold more abundant when cells were grown at low than at high aeration, whereas the flavocytochrome c-fumarate reductase was specifically induced by anaerobic growth on fumarate. When adjusted for the different heme content, the monoheme cytochrome c5 is as abundant as are the small tetraheme cytochrome and the tetraheme fumarate reductase. Published results on regulation of cytochromes from DNA microarrays and 2D-PAGE differ somewhat from our results, emphasizing the importance of multifaceted analyses in proteomics.     

Electron transfer between periplasmic formate dehydrogenase and cytochromes c in Desulfovibrio desulfuricans ATCC 27774

Desulfovibrio spp. are sulfate-reducing organisms characterized by having multiple periplasmic hydrogenases and formate dehydrogenases (FDHs). In contrast to enzymes in most bacteria, these enzymes do not reduce directly the quinone pool, but transfer electrons to soluble cytochromes c. Several studies have investigated electron transfer with hydrogenases, but comparatively less is known about FDHs. In this work we conducted experiments to assess potential electron transfer pathways resulting from formate oxidation in Desulfovibrio desulfuricans ATCC 27774. This organism can grow on sulfate and on nitrate, and contains a single soluble periplasmic FDH that includes a cytochrome c (3) like subunit (FdhABC(3)). It has also a unique cytochrome c composition, including two cytochromes c not yet isolated from other species, the split-Soret and nine-heme cytochromes, besides a tetraheme type I cytochrome c (3) (TpIc (3)). The FDH activity and cytochrome composition of cells grown with lactate or formate and nitrate or sulfate were determined, and the electron transfer between FDH and these cytochromes was investigated. We studied also the reduction of the Dsr complex and of the monoheme cytochrome c-553, previously proposed to be the physiological partner of FDH. FdhABC(3) was able to reduce the c-553, TpIc (3), and split-Soret cytochromes with a high rate. For comparison, the same experiments were performed with the [NiFe] hydrogenase from the same organism. This study shows that FdhABC(3) can directly reduce the periplasmic cytochrome c network, feeding electrons into several alternative metabolic pathways, which explains the advantage of not having an associated membrane subunit.     

Characterization of the NapGH quinol dehydrogenase complex involved in Wolinella succinogenes nitrate respiration

Nitrate respiration catalysed by the ε-proteobacterium Wolinella succinogenes relies on the NapAGHBFLD system that comprises periplasmic nitrate reductase (NapA) and various other Nap proteins required for electron transport from menaquinol to NapA or maturation of Nap components. The W. succinogenes Nap system is unusual as electron transfer to NapA was shown previously to depend on both subunits of the predicted menaquinol dehydrogenase complex NapGH but did not require a cytochrome c of the NapC/NrfH family. Nonetheless, minor residual growth by nitrate respiration was observed in napG and napH gene inactivation mutants. Here, the question is addressed whether alternative membrane-bound menaquinol dehydrogenases, like NrfH and NosGH, involved in nitrite or N₂O reduction systems, are able to functionally replace NapGH. The phenotypes of various gene deletion mutants as well as strains expressing chimeric nap / nos operons demonstrate that NosH is able to donate electrons to the respiratory chain of nitrate respiration at a physiologically relevant rate, whereas NrfH and NosG are not. The iron-sulphur protein NapG was shown to form a complex with NapH in the membrane but was detected in the periplasmic cell fraction in the absence of NapH. Likewise, NosH is able to bind NapG. Each of the eight poly-cysteine motifs present in either NapG or NapH was shown to be essential for nitrate respiration. The NapG homologue NosG could not substitute for NapG, even after adjusting the cysteine spacing to that of NapG, implying that NapG and NosG are specific adapter proteins that channel electrons into either the Nap or Nos system. The current model on the structure and function of the NapGH menaquinol dehydrogenase complex is presented and the composition of the electron transport chains that deliver electrons to periplasmic reductases for either nitrate, nitrite or N₂O is discussed.     

Sulfate respiration in Desulfovibrio vulgaris Hildenborough. Structure of the 16-heme cytochrome c HmcA AT 2.5-A resolution and a view of its role in transmembrane electron transfer

The crystal structure of the high molecular mass cytochrome c HmcA from Desulfovibrio vulgaris Hildenborough is described. HmcA contains the unprecedented number of sixteen hemes c attached to a single polypeptide chain, is associated with a membrane-bound redox complex, and is involved in electron transfer from the periplasmic oxidation of hydrogen to the cytoplasmic reduction of sulfate. The structure of HmcA is organized into four tetraheme cytochrome c(3)-like domains, of which the first is incomplete and contains only three hemes, and the final two show great similarity to the nine-heme cytochrome c from Desulfovibrio desulfuricans. An isoleucine residue fills the vacant coordination space above the iron atom in the five-coordinated high-spin Heme 15. The characteristics of each of the tetraheme domains of HmcA, as well as its surface charge distribution, indicate this cytochrome has several similarities with the nine-heme cytochrome c and the Type II cytochrome c(3) molecules, in agreement with their similar genetic organization and mode of reactivity and further support an analogous physiological function for the three cytochromes. Based on the present structure, the possible electron transfer sites between HmcA and its redox partners (namely Type I cytochrome c(3) and other proteins of the Hmc complex), as well as its physiological role, are discussed.     

Enzymology and bioenergetics of respiratory nitrite ammonification

Nitrite is widely used by bacteria as an electron acceptor under anaerobic conditions. In respiratory nitrite ammonification an electrochemical proton potential across the membrane is generated by electron transport from a non-fermentable substrate like formate or H(2) to nitrite. The corresponding electron transport chain minimally comprises formate dehydrogenase or hydrogenase, a respiratory quinone and cytochrome c nitrite reductase. The catalytic subunit of the latter enzyme (NrfA) catalyzes nitrite reduction to ammonia without liberating intermediate products. This review focuses on recent progress that has been made in understanding the enzymology and bioenergetics of respiratory nitrite ammonification. High-resolution structures of NrfA proteins from different bacteria have been determined, and many nrf operons sequenced, leading to the prediction of electron transfer pathways from the quinone pool to NrfA. Furthermore, the coupled electron transport chain from formate to nitrite of Wolinella succinogenes has been reconstituted by incorporating the purified enzymes into liposomes. The NrfH protein of W. succinogenes, a tetraheme c-type cytochrome of the NapC/NirT family, forms a stable complex with NrfA in the membrane and serves in passing electrons from menaquinol to NrfA. Proteins similar to NrfH are predicted by open reading frames of several bacterial nrf gene clusters. In gamma-proteobacteria, however, NrfH is thought to be replaced by the nrfBCD gene products. The active site heme c group of NrfA proteins from different bacteria is covalently bound via the cysteine residues of a unique CXXCK motif. The lysine residue of this motif serves as an axial ligand to the heme iron thus replacing the conventional histidine residue. The attachment of the lysine-ligated heme group requires specialized proteins in W. succinogenes and Escherichia coli that are encoded by accessory nrf genes. The proteins predicted by these genes are unrelated in the two bacteria but similar to proteins of the respective conventional cytochrome c biogenesis systems.     

NapF is a cytoplasmic iron-sulfur protein required for Fe-S cluster assembly in the periplasmic nitrate reductase

The periplasmic nitrate reductase (Nap) is wide-spread in proteobacteria. NapA, the nitrate reductase catalytic subunit, contains a Mo-bisMGD cofactor and one [4Fe-4S] cluster. The nap gene clusters in many bacteria, including Rhodobacter sphaeroides DSM158, contain an napF gene, disruption of which drastically decreases both in vitro and in vivo nitrate reductase activities. In spite its importance in the Nap system, NapF has never been characterized biochemically, and its role remains unknown. The NapF protein has four polycysteine clusters that suggest that it is an iron-sulfur-containing protein. In the present study, a His(6)-tagged NapF protein was overproduced in Escherichia coli and purified anaerobically. The purified NapF protein was used to obtain polyclonal antibodies raised in rabbit, and cellular fractionation of R. sphaeroides followed by immunoprobing with anti-NapF antibodies revealed that the native NapF protein is located in the cytoplasm. This contrasts with the periplasmic location of the mature NapA. However, NapA could not be detected in an isogenic napF(-) strain of R. sphaeroides. The His(6)-tagged NapF protein displayed spectral properties indicative of Fe-S clusters, but these features were rapidly lost, suggesting cluster lability. However, reconstitution of the Fe-S centers into the apo-NapF protein was achieved in the presence of Azotobacter vinelandii cysteine desulfurase (NifS), and this allowed the recovery of nitrate reductase activity in NapA protein that had previously been treated with 2,2'-dipyridyl to remove the [4Fe-4S] cluster. This activity was not recovered in the absence of NapF. Taking into account the cytoplasmic localization of NapF, the presence of labile Fe-S clusters in the protein, the napF(-) strain phenotype, and the NapF-dependent reactivation of the 2,2'-dipyridyl-treated NapA, we propose a role for NapF in assembling the [4Fe-4S] center of the catalytic subunit NapA.     

Nonaheme cytochrome c, a new physiological electron acceptor for [Ni,Fe] hydrogenase in the sulfate-reducing bacterium Desulfovibrio desulfuricans Essex: primary sequence, molecular parameters, and redox properties

A nonaheme cytochrome c was purified to homogeneity from the soluble and the membrane fractions of the sulfate-reducing bacterium Desulfovibrio desulfuricans Essex. The gene encoding for the protein was cloned and sequenced. The primary structure of the multiheme protein was highly homologous to that of the nonaheme cytochrome c from D. desulfuricans ATCC 27774 and to that of the 16-heme HmcA protein from Desulfovibrio vulgaris Hildenborough. The analysis of the sequence downstream of the gene encoding for the nonaheme cytochrome c from D. desulfuricans Essex revealed an open reading frame encoding for an HmcB homologue. This operon structure indicated the presence of an Hmc complex in D. desulfuricans Essex, with the nonaheme cytochrome c replacing the 16-heme HmcA protein found in D. vulgaris. The molecular and spectroscopic parameters of nonaheme cytochrome c from D. desulfuricans Essex in the oxidized and reduced states were analyzed. Upon reduction, the pI of the protein changed significantly from 8.25 to 5.0 when going from the Fe(III) to the Fe(II) state. Such redox-induced changes in pI have not been reported for cytochromes thus far; most likely they are the result of a conformational rearrangement of the protein structure, which was confirmed by CD spectroscopy. The reactivity of the nonaheme cytochrome c toward [Ni,Fe] hydrogenase was compared with that of the tetraheme cytochrome c(3); both the cytochrome c(3) and the periplasmic [Ni,Fe] hydrogenase originated from D. desulfuricans Essex. The nonaheme protein displayed an affinity and reactivity toward [Ni,Fe] hydrogenase [K(M) = 20.5 +/- 0.9 microM; v(max) = 660 +/- 20 nmol of reduced cytochrome min(-1) (nmol of hydrogenase)(-1)] similar to that of cytochrome c(3) [K(M) = 12.6 +/- 0.7 microM; v(max) = 790 +/- 30 nmol of reduced cytochrome min(-1) (nmol of hydrogenase)(-1)]. This shows that nonaheme cytochrome c is a competent physiological electron acceptor for [Ni,Fe] hydrogenase.     

The membrane-bound tetrahaem c-type cytochrome CymA interacts directly with the soluble fumarate reductase in Shewanella

Shewanella spp. demonstrate great variability in the use of terminal electron acceptors in anaerobic respiration; these include nitrate, fumarate, DMSO, trimethylamine oxide, sulphur compounds and metal oxides. These pathways open up possible applications in bioremediation. The wide variety of respiratory substrates for Shewanella is correlated with the evolution of several multi-haem membrane-bound, periplasmic and outer-membrane c-type cytochromes. The 21 kDa c-type cytochrome CymA of the freshwater strain Shewanella oneidensis MR-1 has an N-terminal membrane anchor and a globular tetrahaem periplasmic domain. According to sequence alignments, CymA is a member of the NapC/NirT family. This family of redox proteins is responsible for electron transfer from the quinone pool to periplasmic and outer-membrane-bound reductases. Prior investigations have shown that the absence of CymA results in loss of the ability to respire with Fe(III), fumarate and nitrate, indicating that CymA is involved in electron transfer to several terminal reductases. Here we describe the expression, purification and characterization of a soluble, truncated CymA ('CymA). Potentiometric studies suggest that there are two pairs of haems with potentials of -175 and -261 mV and that 'CymA is an efficient electron donor for the soluble fumarate reductase, flavocytochrome c(3).     

Purification and characterization of cytochrome c3, ferredoxin, and rubredoxin isolated from Desulfovibrio desulfuricans Norway.

Different electron carriers of the non-desulfoviridin-containing, sulfate-reducing bacterium Desulfovibrio desulfuricans (Norway strain) have been studied. Two nonheme iron proteins, ferredoxin and rubredoxin, have been purified. This ferredoxin contains four atoms of non-heme iron and acid-labile sulfur and six residues of cysteine per molecule. Its amino acid composition suggests that it is homologous with the other Desulfovibrio ferredoxins. The rubredoxin is also an acidic protein of 6,000 molecular weight and contains one atom of iron and four cysteine residues per molecule. The amino acid composition and molecular weight of the cytochrome c3 from D. desulfuricans (strain Norway 4) are reported. Its spectral properties are very similar to those of the other cytochromes c3 (molecular weight, 13,000) of Desulfovibrio and show that it contains four hemes per molecule. This cytochrome has a very low redox potential and acts as a carrier in the coupling of hydrogenase and thiosulfate reductase in extracts of Desulfovibrio gigas and Desulfovibrio desulfuricans (Norway strain) in contrast to D. gigas cytochrome c3 (molecular weight, 13,000). A comparison of the activities of the cytochrome c3 (molecular weight, 13,000) of D. gigas and that of D. desulfuricans in this reaction suggests that these homologous proteins can have different specificity in the electron transfer chain of these bacteria.     

The haem-copper oxygen reductase of Desulfovibrio vulgaris contains a dihaem cytochrome c in subunit II

The genome of the sulphate reducing bacterium Desulfovibrio vulgaris Hildenborough, still considered a strict anaerobe, encodes two oxygen reductases of the bd and haem-copper types. The haem-copper oxygen reductase deduced amino acid sequence reveals that it is a Type A2 enzyme, which in its subunit II contains two c-type haem binding motifs. We have characterized the cytochrome c domain of subunit II and confirmed the binding of two haem groups, both with Met-His iron coordination. Hence, this enzyme constitutes the first example of a ccaa3 haem-copper oxygen reductase. The expression of D. vulgaris haem-copper oxygen reductase was found to be independent of the electron donor and acceptor source and is not altered by stress factors such as oxygen exposure, nitrite, nitrate, and iron; therefore the haem-copper oxygen reductase seems to be constitutive. The KCN sensitive oxygen reduction by D. vulgaris membranes demonstrated in this work indicates the presence of an active haem-copper oxygen reductase. D. vulgaris membranes perform oxygen reduction when accepting electrons from the monohaem cytochrome c553, thus revealing the first possible electron donor to the terminal oxygen reductase of D. vulgaris. The physiological implication of the presence of the oxygen reductase in this organism is discussed.     

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.