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.     

A directional electron transfer regulator based on heme-chain architecture in the small tetraheme cytochrome c from Shewanella oneidensis

The macroscopic and microscopic redox potentials of the four hemes of the small tetraheme cytochrome c from Shewanella oneidensis were determined. The microscopic redox potentials show that the order of reduction is from hemes in the C-terminal domain (hemes 3 and 4) to the N-terminal domain (heme 1), demonstrating the polarization of the tetraheme chain during reduction. This makes heme 4 the most efficient electron delivery site. Furthermore, multi-step reduction of other redox centers through either heme 4 or heme 3 is shown to be possible. This has provided new insights into the two-electron reduction of the flavin in the homologous flavocytochrome c-fumarate reductase.     

Crystal structures at atomic resolution reveal the novel concept of "electron-harvesting" as a role for the small tetraheme cytochrome c

The genus Shewanella produces a unique small tetraheme cytochrome c that is implicated in the iron oxide respiration pathway. It is similar in heme content and redox potential to the well known cytochromes c(3) but related in structure to the cytochrome c domain of soluble fumarate reductases from Shewanella sp. We report the crystal structure of the small tetraheme cytochrome c from Shewanella oneidensis MR-1 in two crystal forms and two redox states. The overall fold and heme core are surprisingly different from the soluble fumarate reductase structures. The high resolution obtained for an oxidized orthorhombic crystal (0.97 A) revealed several flexible regions. Comparison of the six monomers in the oxidized monoclinic space group (1.55 A) indicates flexibility in the C-terminal region containing heme IV. The reduced orthorhombic crystal structure (1.02 A) revealed subtle differences in the position of several residues, resulting in decreased solvent accessibility of hemes and the withdrawal of a positive charge from the molecular surface. The packing between monomers indicates that intermolecular electron transfer between any heme pair is possible. This suggests there is no unique site of electron transfer on the surface of the protein and that electron transfer partners may interact with any of the hemes, a process termed "electron-harvesting." This optimizes the efficiency of intermolecular electron transfer by maximizing chances of productive collision with redox partners.     

Sequence of the gene encoding flavocytochrome c from Shewanella putrefaciens: a tetraheme flavoenzyme that is a soluble fumarate reductase related to the membrane-bound enzymes from other bacteria.

Flavocytochrome c from the Gram-negative, food-spoiling bacterium Shewanella putrefaciens is a soluble, periplasmic fumarate reductase. We have isolated the gene encoding flavocytochrome c and determined the complete DNA sequence. The predicted amino acid sequence indicates that flavocytochrome c is synthesized with an N-terminal secretory signal sequence of 25 amino acid residues. The mature protein contains 571 amino acid residues and consists of an N-terminal cytochrome domain, of about 117 residues, with four heme attachment sites typical of c-type cytochromes and a C-terminal flavoprotein domain of about 454 residues that is clearly related to the flavoprotein subunits of fumarate reductases and succinate dehydrogenases from bacterial and other sources. A second reading frame that may be cotranscribed with the flavocytochrome c gene exhibits some similarity with the 13-kDa membrane anchor subunit of Escherichia coli fumarate reductase. The sequence of the flavoprotein domain demonstrates an even closer relationship with the product of the yeast OSM1 gene, mutations in which result in sensitivity to high osmolarity. These findings are discussed in relation to the function of flavocytochrome c.     

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).     

Structure and mechanism of the flavocytochrome c fumarate reductase of Shewanella putrefaciens MR-1

Fumarate respiration is one of the most widespread types of anaerobic respiration. The soluble fumarate reductase of Shewanella putrefaciens MR-1 is a periplasmic tetraheme flavocytochrome c. The crystal structures of the enzyme were solved to 2.9 A for the uncomplexed form and to 2.8 A and 2.5 A for the fumarate and the succinate-bound protein, respectively. The structures reveal a flexible capping domain linked to the FAD-binding domain. A catalytic mechanism for fumarate reduction based on the structure of the complexed protein is proposed. The mechanism for the reverse reaction is a model for the homologous succinate dehydrogenase (complex II) of the respiratory chain. In flavocytochrome c fumarate reductase, all redox centers are in van der Waals contact with one another, thus providing an efficient conduit of electrons from the hemes via the FAD to fumarate.     

The solution structure of a tetraheme cytochrome from Shewanella frigidimarina reveals a novel family structural motif

The bacteria belonging to the genus Shewanella are facultative anaerobes that utilize a variety of terminal electron acceptors which includes soluble and insoluble metal oxides. The tetraheme c-type cytochrome isolated during anaerobic growth of Shewanella frigidimarina NCIMB400 ( Sfc) contains 86 residues and is involved in the Fe(III) reduction pathways. Although the functional properties of Sfc redox centers are quite well described, no structures are available for this protein. In this work, we report the solution structure of the reduced form of Sfc. The overall fold is completely different from those of the tetraheme cytochromes c 3 and instead has similarities with the tetraheme cytochrome recently isolated from Shewanella oneidensis ( Soc). Comparison of the tetraheme cytochromes from Shewanella shows a considerable diversity in their primary structure and heme reduction potentials, yet they have highly conserved heme geometry, as is the case for the family of tetraheme cytochromes isolated from Desulfovibrio spp.     

Nitrate reduction by Desulfovibrio desulfuricans: a periplasmic nitrate reductase system that lacks NapB, but includes a unique tetraheme c-type cytochrome, NapM

Many sulphate reducing bacteria can also reduce nitrite, but relatively few isolates are known to reduce nitrate. Although nitrate reductase genes are absent from Desulfovibrio vulgaris strain Hildenborough, for which the complete genome sequence has been reported, a single subunit periplasmic nitrate reductase, NapA, was purified from Desulfovibrio desulfuricans strain 27774, and the structural gene was cloned and sequenced. Chromosome walking methods have now been used to determine the complete sequence of the nap gene cluster from this organism. The data confirm the absence of a napB homologue, but reveal a novel six-gene organisation, napC-napM-napA-napD-napG-napH. The NapC polypeptide is more similar to the NrfH subgroup of tetraheme cytochromes than to NapC from other bacteria. NapM is predicted to be a tetra-heme c-type cytochrome with similarity to the small tetraheme cytochromes from Shewanella oneidensis. The operon is located close to a gene encoding a lysyl-tRNA synthetase that is also found in D. vulgaris. We suggest that electrons might be transferred to NapA either from menaquinol via NapC, or from other electron donors such as formate or hydrogen via the small tetraheme cytochrome, NapM. We also suggest that, despite the absence of a twin-arginine targeting sequence, NapG might be located in the periplasm where it would provide an alternative direct electron donor to NapA.     

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.     

Expression and characterization of the diheme cytochrome c subunit of the cytochrome bc complex in Heliobacterium modesticaldum

Heliobacterium modesticaldum is a Gram-positive, anaerobic, anoxygenic photoheterotrophic bacterium. Its cytochrome bc complex (Rieske/cyt b complex) has some similarities to cytochrome b(6)f complexes from cyanobacteria and chloroplasts, and also shares some characteristics of typical bacterial cytochrome bc(1) complexes. One of the unique factors of the heliobacterial cytochrome bc complex is the presence of a diheme cytochrome c instead of the monoheme cytochrome f in the cytochrome b(6)f complex or the monoheme cytochrome c(1) in the bc(1) complex. To understand the structure and function of this diheme cytochrome c protein, we expressed the N-terminal transmembrane-helix-truncated soluble H. modesticaldum diheme cytochrome c in Escherichia coli. This 25kDa recombinant protein possesses two c-type hemes, confirmed by mass spectrometry and a variety of biochemical techniques. Sequence analysis of the H. modesticaldum diheme cytochrome c indicates that it may have originated from gene duplication and subsequent gene fusion, as in cytochrome c(4) proteins. The recombinant protein exhibits a single redox midpoint potential of +71mV versus NHE, which indicates that the two hemes have very similar protein environments.     

Process of maturation of tetraheme cytochrome c3 in a Shewanella expression system

The process of maturation of multiheme proteins is not yet well known, while that of monoheme ones has been relatively well investigated. Two kinds of partly unfolded tetraheme cytochrome c3 were obtained on overexpression in Shewanella oneidensis TSP-C. These proteins were characterized by circular dichroism and nuclear magnetic resonance spectroscopy. It turned out that the tetraheme architecture, and the fifth and sixth ligand coordination are almost mature, while some parts of the polypeptide are unfolded. The unfolded residues are mainly located in the helix-rich region including heme attachment and axial ligand sites. This suggests that the formation of the heme architecture, coordination of axial ligands and helix formation should be coupled with each other. While the former two can take place automatically, the helix formation would need help by a chaperone-like function in the cytochrome c maturation (Ccm) machinery. It must be working in sulphate-reducing bacteria. The Ccm machinery in S. oneidensis is likely insufficient to help the maturation of proteins with cyclic heme architectures. This is the first report providing an insight into the process of maturation of tetraheme cytochrome c3.     

Covalent structure of the diheme cytochrome subunit and amino-terminal sequence of the flavoprotein subunit of flavocytochrome c from Chromatium vinosum.

The complete sequence of the 21-kDa cytochrome subunit of the flavocytochrome c (FC) from the purple phototrophic bacterium Chromatium vinosum has been determined to be as follows: EPTAEMLTNNCAGCHG THGNSVGPASPSIAQMDPMVFVEVMEGFKSGEIAS TIMGRIAKGYSTADFEKMAGYFKQQTYQPAKQSF DTALADTGAKLHDKYCEKCHVEGGKPLADEEDY HILAGQWTPYLQYAMSDFREERRPMEKKMASKL RELLKAEGDAGLDALFAFYASQQ. The sequence is the first example of a diheme cytochrome in a flavocytochrome complex. Although the locations of the heme binding sites and the heme ligands suggest that the cytochrome subunit is the result of gene doubling of a type I cytochrome c, as found with Azotobacter cytochrome c4, the extremely low similarity of only 7% between the two halves of the Chromatium FC heme subunit rather suggests that gene fusion is at the evolutionary origin of this cytochrome. The two halves also require a single residue internal deletion for alignment. The first half of the Chromatium FC heme subunit is 39% similar to the monoheme subunit of the FC from the green phototrophic bacterium Chlorobium thiosulfatophilum, but the second half is only 9% similar to the Chlorobium subunit. The N-terminal sequence of the Chromatium FC flavin subunit was determined up to residue 41 as AGRKVVVVGGGTGGATAAKYIKLADPSIEVTLIEP NTKYYT. It shows more similarity to the Chlorobium FC flavin subunit (60%) than do the two heme subunits. The N terminus of the flavin subunit is homologous to a number of flavoproteins, including succinate dehydrogenase, glutathione reductase, and monamine oxidase. There is no obvious homology to the Pseudomonas putida FC flavin subunit, which suggests that the two types of flavocytochrome c arose by convergent evolution. This is consistent with the dissimilar enzyme activities of FC as sulfide dehydrogenase in the phototrophic bacteria and as p-cresol methylhydroxylase in Pseudomonas. We also present a sequence "fingerprint" pattern for the recognition of FAD-binding proteins which is an extended version of the consensus sequence previously presented (Wierenga, R. K., Terpstra, P., and Hol, W. G. J. (1986) J. Mol. Biol. 187, 101-107) for nucleotide binding sites.     

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.     

The tetraheme cytochrome CymA is required for anaerobic respiration with dimethyl sulfoxide and nitrite in Shewanella oneidensis

The tetraheme c-type cytochrome, CymA, from Shewanella oneidensis MR-1 has previously been shown to be required for respiration with Fe(III), nitrate, and fumarate [Myers, C. R., and Myers, J. M. (1997) J. Bacteriol. 179, 1143-1152]. It is located in the cytoplasmic membrane where the bulk of the protein is exposed to the periplasm, enabling it to transfer electrons to a series of redox partners. We have expressed and purified a soluble derivative of CymA (CymA(sol)) that lacks the N-terminal membrane anchor. We show here, by direct measurements of electron transfer between the purified proteins, that CymA(sol) efficiently reduces S. oneidensis fumarate reductase. This indicates that no further proteins are required for electron transfer between the quinone pool and fumarate if we assume direct reduction of CymA by quinols. By expressing CymA(sol) in a mutant lacking CymA, we have shown that this soluble form of the protein can complement the defect in fumarate respiration. We also demonstrate that CymA is essential for growth with DMSO (dimethyl sulfoxide) and for reduction of nitrite, implicating CymA in at least five different electron transfer pathways in Shewanella.     

Characterization of three soluble c-type cytochromes isolated from soybean root nodule bacteroids of Bradyrhizobium japonicum strain CC705

Three soluble, low molecular mass cytochromes c (Mr 8000-15,000) were isolated and purified from soybean root nodule bacteroids of Bradyrhizobium japonicum strain CC705. On the basis of their alpha: absorbance peaks in the reduced forms, they were named cytochromes c550, c552 and c555. Cytochrome c552 reacted very fast, c555 very slowly and c550 not at all with carbon monoxide. The complete amino acid sequence (73 residues) of cytochrome c552 was established which identifies it as a monoheme, class I cytochrome c with some remote similarity to the cytochrome c6 family.     

Comparison of cytochromes from anaerobically and aerobically grown cells of Pseudomonas perfectomarinus.

Pseudomonas perfectomarinus (ATCC 14405) is a facultative anaerobe capable of either oxygen respiration or anaerobic nitrate respiration, i.e., denitrification. A comparative study of the electron transfer components of cells revealed five c-type cytochromes and cytochrome cd in the soluble fraction from anaerobically grown cells and four c-type cytochromes in the soluble fraction from aerobically grown cells. Purification procedures yielded three c-type cytochromes (designated c-551, c-554, and acidic c-type) from both kinds of cells as indicated by similarities in absorption spectra, molecular weight, and electrophoretic mobility. Cytochrome cd, a diheme c-type cytochrome (cytochrome c-552), and a split-alpha c-type cytochrome were recovered only from anaerobically grown cells. A c-type cytochrome with a low ratio of alpha to beta absorption peak heights was uniquely present in the aerobically grown cells. Liquid N2 temperature absorption spectroscopy on the membrane fraction from anaerobically grown cells revealed residual cytochrome cd as well as differences in the relative amounts of c-type and b-type cytochromes in membranes prepared from cells grown under the two different conditions.     

Cloning and characterization of sulfite dehydrogenase, two c-type cytochromes, and a flavoprotein of Paracoccus denitrificans GB17: essential role of sulfite dehydrogenase in lithotrophic sulfur oxidation.

A 13-kb genomic region of Paracoccus dentrificans GB17 is involved in lithotrophic thiosulfate oxidation. Adjacent to the previously reported soxB gene (C. Wodara, S. Kostka, M. Egert, D. P. Kelly, and C. G. Friedrich, J. Bacteriol. 176:6188-6191, 1994), 3.7 kb were sequenced. Sequence analysis revealed four additional open reading frames, soxCDEF. soxC coded for a 430-amino-acid polypeptide with an Mr of 47,339 that included a putative signal peptide of 40 amino acids (Mr of 3,599) with a RR motif present in periplasmic proteins with complex redox centers. The mature soxC gene product exhibited high amino acid sequence similarity to the eukaryotic molybdoenzyme sulfite oxidase and to nitrate reductase. We constructed a mutant, GBsoxC delta, carrying an in-frame deletion in soxC which covered a region possibly coding for the molybdenum cofactor binding domain. GBsoxC delta was unable to grow lithoautotrophically with thiosulfate but grew well with nitrate as a nitrogen source or as an electron acceptor. Whole cells and cell extracts of mutant GBsoxC delta contained 10% of the thiosulfate-oxidizing activity of the wild type. Only a marginal rate of sulfite-dependent cytochrome c reduction was observed from cell extracts of mutant GBsoxC delta. These results demonstrated that sulfite dehydrogenase was essential for growth with thiosulfate of P. dentrificans GB17. soxD coded for a periplasmic diheme c-type cytochrome of 384 amino acids (Mr of 39,983) containing a putative signal peptide with an Mr of 2,363. soxE coded for a periplasmic monoheme c-type cytochrome of 236 amino acids (Mr of 25,926) containing a putative signal peptide with an Mr of 1,833. SoxD and SoxE were highly identical to c-type cytochromes of P. denitrificans and other organisms. soxF revealed an incomplete open reading frame coding for a peptide of 247 amino acids with a putative signal peptide (Mr of 2,629). The deduced amino acid sequence of soxF was 47% identical and 70% similar to the sequence of the flavoprotein of flavocytochrome c of Chromatium vinosum, suggesting the involvement of the flavoprotein in thiosulfate oxidation of P. denitrificans GB17.