Multiheme C-type Cytochromes Research Articles

Molecular details of multielectron transfer: the case of multiheme cytochromes from metal respiring organisms

Shewanella are facultative anaerobic bacteria of remarkable respiratory versatility that includes the dissimilatory reduction of metal ores. They contain a large number of multiheme c-type cytochromes that play a significant role in various anaerobic respiratory processes. Of all the cytochromes found in Shewanella, only the two most abundant periplasmic cytochromes, the small tetraheme cytochrome (STC) and flavocytochrome c(3) (Fcc(3)) have been structurally characterized. For these two proteins the molecular bases for their redox properties were determined using spectroscopic methods based on paramagnetic NMR, that allow the contribution of specific hemes to be discriminated. In this perspective these results are reviewed in the context of the continuing effort to understand the molecular mechanisms of electron transfer in the respiratory chains of these organisms.

Electron transfer at the microbe–mineral interface: a grand challenge in biogeochemistry

The interplay between microorganisms and minerals is a complex and dynamic process that has sculpted the geosphere for nearly the entire history of the Earth. The work of Dr Terry Beveridge and colleagues provided some of the first insights into metal-microbe and mineral-microbe interactions and established a foundation for subsequent detailed investigations of interactions between microorganisms and minerals. Beveridge also envisioned that interdisciplinary approaches and teams would be required to explain how individual microbial cells interact with their immediate environment at nano- or microscopic scales and that through such approaches and using emerging technologies that the details of such interactions would be revealed at the molecular level. With this vision as incentive and inspiration, a multidisciplinary, collaborative team-based investigation was initiated to probe the process of electron transfer (ET) at the microbe-mineral interface. The grand challenge to this team was to address the hypothesis that multiheme c-type cytochromes of dissimilatory metal-reducing bacteria localized to the cell exterior function as the terminal reductases in ET to Fe(III) and Mn(IV) oxides. This question has been the subject of extensive investigation for years, yet the answer has remained elusive. The team involves an integrated group of experimental and computational capabilities at US Department of Energy's Environmental Molecular Sciences Laboratory, a national scientific user facility, as the collaborative focal point. The approach involves a combination of in vitro and in vivo biologic and biogeochemical experiments and computational analyses that, when integrated, provide a conceptual model of the ET process. The resulting conceptual model will be evaluated by integrating and comparing various experimental, i.e. in vitro and in vivo ET kinetics, and theoretical results. Collectively, the grand challenge will provide a detailed view of how organisms engage with mineral surfaces to exchange energy and electron density as required for life function.

Isotopic labeling of c-type multiheme cytochromes overexpressed in E. coli

Progresses made in bacterial genome sequencing show a remarkable profusion of multiheme c-type cytochromes in many bacteria, highlighting the importance of these proteins in different cellular events. However, the characterization of multiheme cytochromes has been significantly retarded by the numerous experimental challenges encountered by researchers who attempt to overexpress these proteins, especially if isotopic labeling is required. Here we describe a methodology for isotopic labeling of multiheme cytochromes c overexpressed in Escherichia coli, using the triheme cytochrome PpcA from Geobacter sulfurreducens as a model protein. By combining different strategies previously described and using E. coli cells containing the gene coding for PpcA and the cytochrome c maturation gene cluster, an experimental labeling methodology was developed that is based on two major aspects: (i) use of a two-step culture growth procedure, where cell growth in rich media was followed by transfer to minimal media containing 15N-labeled ammonium chloride, and (ii) incorporation of the heme precursor delta-aminolevulinic acid in minimal culture media. The yields of labeled protein obtained were comparable to those obtained for expression of PpcA in rich media. Proper protein folding and labeling were confirmed by UV–visible and NMR spectroscopy. To our knowledge, this is the first report of a recombinant multiheme cytochrome labeling and it represents a major breakthrough for functional and structural studies of multiheme cytochromes.

Redox-linked conformational changes of a multiheme cytochrome from Geobacter sulfurreducens

Multiheme c-type cytochromes from members of the Desulfovibrionacea and Geobactereacea families play crucial roles in the bioenergetics of these microorganisms. Thermodynamic studies using NMR and visible spectroscopic techniques on tetraheme cytochromes c 3 isolated from Desulfovibrio spp. and more recently on a triheme cytochrome from Geobacter sulfurreducens showed that the properties of each redox centre are modulated by the neighbouring redox centres enabling these proteins to perform energy transduction and thus contributing to cellular energy conservation. Electron/proton transfer coupling relies on redox-linked conformational changes that were addressed for some multiheme cytochromes from the comparison of protein structure of fully reduced and fully oxidised forms. In this work, we identify for the first time in a multiheme cytochrome the simultaneous presence of two different conformations in solution. This was achieved by probing the different oxidation stages of a triheme cytochrome isolated from G. sulfurreducens using 2D-NMR techniques. The results presented here will be the foundations to evaluate the modulation of the redox centres properties by conformational changes that occur during the reoxidation of a multiheme protein.

Overexpression of Multi-Heme C-type Cytochromes

ABSTRACT-Because they contain covalently attached hemes, c-type cytochromes, especially those with multi-heme, are difficult to over-express. The gram negative bacterium Shewanella oneidensis MR-1 has been successfully used for over-expression of multi-heme c-type cytochromes...

The Crystal Structure of the Hexadeca-Heme Cytochrome Hmc and a Structural Model of Its Complex with Cytochrome c3

Sulfate-reducing bacteria contain a variety of multi-heme c-type cytochromes. The cytochrome of highest molecular weight (Hmc) contains 16 heme groups and is part of a transmembrane complex involved in the sulfate respiration pathway. We present the 2.42 Å resolution crystal structure of the Desulfovibrio vulgaris Hildenborough cytochrome Hmc and a structural model of the complex with its physiological electron transfer partner, cytochrome c 3, obtained by NMR restrained soft-docking calculations. The Hmc is composed of three domains, which exist independently in different sulfate-reducing species, namely cytochrome c 3, cytochrome c 7, and Hcc. The complex involves the last heme at the C-terminal region of the V-shaped Hmc and heme 4 of cytochrome c 3, and represents an example for specific cytochrome-cytochrome interaction.

Modification of heme c binding motifs in the small subunit (NrfH) of the Wolinella succinogenes cytochrome c nitrite reductase complex

The two multiheme c-type cytochromes NrfH and NrfA form a membrane-bound complex that catalyzes menaquinol oxidation by nitrite during respiratory nitrite ammonification of Wolinella succinogenes. Each cysteine residue of the four NrfH heme c binding motifs was individually replaced by serine. Of the resulting eight W. succinogenes mutants, only one is able to grow by nitrite respiration although its electron transport activity from formate to nitrite is decreased. NrfH from this mutant was shown by matrix-assisted laser desorption/ionization mass spectrometry to carry four covalently bound heme groups like wild-type NrfH indicating that the cytochrome c biogenesis system II organism W. succinogenes is able to attach heme to an SXXCH motif.

Purification and Magneto-optical Spectroscopic Characterization of Cytoplasmic Membrane and Outer Membrane Multiheme c-Type Cytochromes from Shewanella frigidimarina NCIMB400

Two membranous c-type cytochromes from the Fe(III)-respiring bacterium Shewanella frigidimarina NCIMB400, CymA and OmcA, have been purified and characterized by UV-visible, magnetic circular dichroism, and electron paramagnetic resonance spectroscopies. The 20-kDa CymA is a member of the NapC/NirT family of multiheme cytochromes, which are invariably anchored to the cytoplasmic membrane of Gram-negative bacteria, and are postulated to mediate electron flow between quinols and periplasmic redox proteins. CymA was found to contain four low-spin c-hemes, each with bis-His axial ligation, and midpoint reduction potentials of +10, -108, -136, and -229 mV. The 85-kDa OmcA is located at the outer membrane of S. frigidimarina NCIMB400, and as such might function as a terminal reductase via interaction with insoluble Fe(III) substrates. This putative role is supported by the finding that the protein was released into solution upon incubation of harvested intact cells at 25 degrees C, suggesting an attachment to the exterior face of the outer membrane. OmcA was revealed by magneto-optical spectrocopies to contain 10 low-spin bis-His ligated c-hemes, with the redox titer indicating two sets of near iso-potential components centered at -243 and -324 mV.

The Desulfuromonas acetoxidans triheme cytochrome c7 produced in Desulfovibrio desulfuricans retains its metal reductase activity.

Multiheme cytochrome c proteins that belong to class III have been recently shown to exhibit a metal reductase activity, which could be of great environmental interest, especially in metal bioremediation. To get a better understanding of these activities, the gene encoding cytochrome c7 from the sulfur-reducing bacterium Desulfuromonas acetoxidans was cloned from genomic DNA by PCR and expressed in Desulfovibrio desulfuricans G201. The expression system was based on the cyc transcription unit from Desulfovibrio vulgaris Hildenborough and led to the synthesis of holocytochrome c7 when transferred by electrotransformation into the sulfate reducer Desulfovibrio desulfuricans G201. The produced cytochrome was indistinguishable from the protein purified from Desulfuromonas acetoxidans cells with respect to several biochemical and biophysical criteria and exhibited the same metal reductase activities as determined from electrochemical experiments. This suggests that the molecule was correctly folded in the host organism. Desulfovibrio desulfuricans produces functional multiheme c-type cytochromes from bacteria belonging to a different genus and may be considered a suitable host for the heterologous biogenesis of multiheme c-type cytochromes for either structural or engineering studies. This report, which presents the first example of the transformation of a Desulfovibrio desulfuricans strain by electrotransformation, describes work that is the first necessary step of a protein engineering program that aims to specify the structural features that are responsible for the metal reductase activities of multiheme cytochrome c7.

Cloning and sequence of cymA, a gene encoding a tetraheme cytochrome c required for reduction of iron(III), fumarate, and nitrate by Shewanella putrefaciens MR-1.

The cymA gene, which encodes a tetraheme cytochrome c, was cloned from Shewanella putrefaciens MR-1. This gene complemented a mutant which had a TnphoA insertion in cymA and which was deficient in the respiratory reduction of iron(III), nitrate, fumarate, and manganese(IV). The 561-bp nucleotide sequence of cymA encodes a protein of 187 amino acids with a predicted molecular mass of 20.8 kDa. No N-terminal signal sequence was readily apparent; consistent with this, a cytochrome with a size of 21 kDa was detected in the wild type but was absent in the insertional mutant. The cymA gene is transcribed into an mRNA; the major transcript was approximately 790 bases, suggesting that it is not part of a multicistronic operon. This RNA transcript was not detected in the cymA mutant. The CymA protein was found in the cytoplasmic membrane and soluble fraction of MR-1, and it shares partial amino acid sequence homology with multiheme c-type cytochromes from other bacteria. These cytochromes are ostensibly involved in the transfer of electrons from the cytoplasmic membrane to acceptors in the periplasm. The localization of the fumarate and iron(III) reductases to the periplasm and outer membrane of MR-1, respectively, suggests the possibility of a similar electron transfer role for CymA.

Coordination and redox properties of a novel triheme cytochrome from Desulfovibrio vulgaris (Hildenborough)

A high molecular weight multiheme c-type cytochrome from the sulfate-reducing bacterium Desulfovibrio vulgaris (Hildenborough) has been spectroscopically characterized and compared with the tetraheme cytochrome c3. The protein contains a pentacoordinate high-spin heme (gz 6.0) and two hexacoordinate low-spin hemes (gz 2.95, gy 2.27, gx 1.48). From analysis of the g values for the low-spin hemes by the procedure of Blumberg and Peisach (Palmer, 1983) and comparison with with the optical spectra from a variety of c-type cytochromes, it is likely that these low-spin hemes are bound by two histidine residues. The NO derivative displayed typical rhombic EPR features (gx 2.07, gz 2.02, gy 1.99). Addition of azide does not lead to coupling between heme chromophores, but the ligand is accessible to the high-spin heme. The use of a glassy-carbon electrode to perform direct (no promoter) electrochemistry on the cytochrome is illustrated. Differential pulse polarography of the native protein gave two waves with reduction potentials of -59 (5) and -400 (8) mV (versus NHE). The cyanide adduct gave two waves with reduction potentials of -263 (8) and -401 (8) mV. The cytochrome was found to catalyze the reduction of nitrite and hydroxylamine.

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.