Purification and properties of a low-redox-potential tetraheme cytochrome c3 from Shewanella putrefaciens

Abstract

Shewanella putrefaciens is a facultatively anaerobic bacterium in the gamma group of the proteobacteria, capable of utilizing a wide variety of anaerobic electron acceptors. An examination of its cytochrome content revealed the presence of a tetraheme, low-redox-potential (E 0 ‫؍‬ ؊233 mV), cytochrome c-type cytochrome with a molecular mass of 12,120 Da and a pI of 5.8. The electron spin resonance data indicate a bis-histidine coordination of heme groups. Reduction of ferric citrate was accompanied by oxidation of the cytochrome. The biochemical properties suggested that this protein was in the cytochrome c 3 group, which is supported by N-terminal sequence data up to the first heme binding site.The ability of Shewanella putrefaciens to grow with a variety of different electron acceptors including oxygen, nitrate, sulfur, thiosulfate, sulfite, fumarate, trimethylamine-N-oxide (TMAO), and metal ions (10, 11) raises questions about the architecture of the electron transport chain in this versatile organism. In particular, given the range of electron potentials covered by the various electron acceptors it utilizes, the question of which types and the numbers of cytochromes present becomes an interesting one. In this regard, little detailed work has been done on cytochromes from S. putrefaciens. Early work by Obuekwe and Westlake (12) with whole-cell spectra showed that cytochrome c-type cytochromes were present and that both the red cell coloration and cytochrome content were correlated with the presence of iron in the growth medium. Arnold et al. (2) also reported whole-cell spectra and concluded that cytochrome synthesis was induced by growth under conditions of low oxygen tension. Myers and Myers (9) studied the locations of cytochromes in S. putrefaciens, concluding that many c-type cytochromes were present and that they were primarily localized in the outer membrane. Morris et al. (7) resolved nine c-type cytochrome bands by DEAE-Sepharose chromatography. There were two principal components that eluted at 90 mM and 315 mM NaCl, both of which had low redox potentials. In all of the above-cited studies, cytochrome content and type were judged mainly from spectral data, using the intensity of the Soret band after treatment with dithionite, or by heme staining polyacrylamide gels with tetramethylbenzidine (18). In recent and more definitive studies, a large tetraheme flavocytochrome c fumarate reductase was purified from S. putrefaciens (NCIMB400), and the gene sequence was determined (6,13). This enzyme is a 63.8-kDa soluble protein, and unlike the usual membrane-bound fumarate reductase, it contains heme c instead of iron-sulfur centers, and the heme midpoint redox potentials of Ϫ220 and Ϫ320 mV are unusually low. In a recent study, Lies et al. (5) reported that S. putrefaciens produces isoprenoid quinones of three types, with menaquinones and isoprenoid types predominating under aerobic conditions and methylmenaquinone types being present under anaerobic growth conditions. In this study, we report the purificat...

Regulation of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) metabolism in Shewanella halifaxensis HAW-EB4 by terminal electron acceptor and involvement of c-type cytochrome

“…Regulation by TEA of c-type cytochrome production in S. halifaxensis Several strains of Shewanella have been reported to produce c-type cytochromes capable of reducing insoluble metal oxide (Tsapin et al, 1996(Tsapin et al, , 2001Gordon et al, 2000). In the present study, the cytochrome content of S. halifaxensis HAW-EB4 grown under both anaerobic and aerobic conditions was characterized and determined for its potential involvement in RDX metabolism.…”

“…Shewanella are known to produce diverse soluble lowredox-potential multihaem c-type cytochromes (Tsapin et al, 1996(Tsapin et al, , 2001Gordon et al, 2000), and genomes of multiple strains of Shewanella have been sequenced (http:// img.jgi.doe.gov/). Shewanella is thus an ideal model organism to investigate the molecular mechanisms that underlie the TEA-regulated multiple electron-transfer pathways of anaerobic RDX biotransformation via either initial denitration or initial reduction of -N-NO 2 to -N-NO.…”

Expression of a Tetraheme Protein, Desulfovibrio vulgaris Miyazaki F Cytochrome c 3 , in Shewanella oneidensis MR-1

“…However, although expression was obtained, a user-friendly system for protein production has not yet been achieved. Because Shewanella oneidensis MR-1 is known to produce cytochrome c 3 (14), and because it is a facultative aerobe that can be easily and rapidly grown to high cell densities, we reasoned that it might be useful as an expression vehicle, and we report here that the gene for cytochrome c 3 from D. vulgaris Miyazaki F can be expressed under either aerobic or anaerobic conditions in MR-1.…”

“…The system may also have utility for the expression of other multiheme protein genes, because Shewanella produces a variety of c-type multiheme cytochromes of its own (7,14), suggesting that it has a very good heme ligase system. MR-1 is capable of reduction of both elemental sulfur and thiosulate (8), an unusual ability even for many anaerobes, and mutants lacking cytochromes c are unable to reduce these compounds.…”

Chemical and Biological Interactions during Nitrate and Goethite Reduction by Shewanella putrefaciens 200

“…Studies with S. putrefaciens 200 suggest that this inhibition may result from kinetically favorable transport of electrons to NO 3 Ϫ or NO 2 Ϫ , a mechanism suggested by Arnold et al to explain inhibition of Fe(III) reduction by oxygen (3)(4)(5)14). Although the respiratory electron transport chain of S. putrefaciens is known to include a variety of b-and c-type cytochromes and quinones (36,42,55), the sequential arrangement of electron carriers has not been elucidated, and terminal NO 3 Ϫ , NO 2 Ϫ , or Fe(III) reductases have not been fully purified. While the NO 3 Ϫ and NO 2 Ϫ reductases for S. putrefaciens MR-1 have been partially purified by Krause and Nealson (27), MR-1 is a denitrifier, whereas the S. putrefaciens 200 used in our studies primarily reduces NO 3 Ϫ and NO 2 Ϫ to ammonia (Cooper, Coby, and Picardal, unpublished data).…”