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Five-coordinate heme nitrosyl complexes (5cNO) underpin biological heme-NO signal transduction. Bacterial cytochromes c' are some of the few structurally characterized 5cNO proteins, exhibiting a distal to proximal 5cNO transition of relevance to NO sensing. Establishing how 5cNO coordination (distal vs proximal) depends on the heme environment is important for understanding this process. Recent 5cNO crystal structures of Alcaligenes xylosoxidans cytochrome c' (AXCP) and Shewanella frigidimarina cytochrome c' (SFCP) show a basic residue (Arg124 and Lys126, respectively) near the proximal NO binding sites. Using resonance Raman (RR) spectroscopy, we show that structurally characterized 5cNO complexes of AXCP variants and SFCP exhibit a range of ν(NO) (1651-1671 cm(-1)) and ν(FeNO) (519-536 cm(-1)) vibrational frequencies, depending on the nature of the proximal heme pocket and the sample temperature. While the AXCP Arg124 residue appears to have little impact on 5cNO vibrations, the ν(NO) and ν(FeNO) frequencies of the R124K variant are consistent with (electrostatically) enhanced Fe(II) → (NO)π* backbonding. Notably, RR frequencies for SFCP and R124A AXCP are significantly displaced from the backbonding trendline, which in light of recent crystallographic data and density functional theory modeling may reflect changes in the Fe-N-O angle and/or extent of σ-donation from the NO(π*) to the Fe(II) (dz(2)) orbital. For R124A AXCP, correlation of vibrational and crystallographic data is complicated by distal and proximal 5cNO populations. Overall, this study highlights the complex structure-vibrational relationships of 5cNO proteins that allow RR spectra to distinguish 5cNO coordination in certain electrostatic and steric environments.

Shewanella oneidensis MR-1 has the metabolic capacity to grow anaerobically using Fe(III) as a terminal electron acceptor. Growth under these conditions results in the de novo synthesis of a number of periplasmic c-type cytochromes, many of which are multiheme in nature and are thought to be involved in the Fe(III) respiratory process. To begin a biochemical study of these complex cytochromes, the mtrA gene that encodes an approximate 32-kDa periplasmic decaheme cytochrome has been heterologously expressed in Escherichia coli. Co-expression of mtrA with a plasmid that contains cytochrome c maturation genes leads to the assembly of a correctly targeted holoprotein, which covalently binds ten hemes. The recombinant MtrA protein has been characterized by magnetic circular dichroism, which shows that all ten hemes have bis-histidine axial ligation. EPR spectroscopy detected only eight of these hemes, all of which are low spin and provides evidence for a spin-coupled pair of hemes in the oxidized state. Redox titrations of MtrA have been carried out with optical- and EPR-monitored methods, and the hemes are shown to reduce over the potential range -100 to -400 mV. In intact cells of E. coli, MtrA is shown to obtain electrons from the host electron transport chain and pass these onto host oxidoreductases or a range of soluble Fe(III) species. This demonstrates the promiscuous nature of this decaheme cytochrome and its potential to serve as a soluble Fe(III) reductase in intact cells.

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.

Iron-induced flavocytochrome c(3), Ifc(3), from Shewanella frigidimarina NCIMB400, derivatized with a 2-pyridyl disulfide label, self-assembles on gold electrodes as a functional array whose fumarate reductase activity as viewed by direct electrochemistry is indistinguishable from that of Ifc(3) adsorbed on gold or graphite electrodes. The enhanced stability of the labeled protein's array permits analysis at a rotating electrode and limiting catalytic currents fit well to a Michaelis-Menten description of enzyme kinetics with K(M) = 56 +/- 20 microM, pH 7.5, comparable to that obtained in solution assays. At fumarate concentrations above 145 microM cyclic voltammetry shows the catalytic response to contain two features. The position and width of the lower potential component centered on -290 mV and corresponding to a one-electron wave implicates the oxidation state of the lowest potential heme of Ifc(3) as a defining feature in the mechanism of fumarate reduction at high turnover rates. We propose the operation of dual pathways for electron transfer to the active site of Ifc(3) with the lowest potential heme acting as an electron relay on one of these pathways.

Fumarate reductases and succinate dehydrogenases play central roles in the metabolism of eukaryotic and prokaryotic cells. A recent medium resolution structure of the Escherichia coli fumarate reductase (Frd) has revealed the overall organization of the membrane-bound complex. Here we present the first high resolution X-ray crystal structure of a water-soluble bacterial fumarate reductase in an open conformation. This structure reveals a mobile domain that modulates substrate access to the active site and provides new insights into the mechanism of this widespread and important family of FAD-containing respiratory proteins.

A 63.9 kDa periplasmic tetrahaem flavocytochrome c(3), designated Ifc(3), was found to be expressed in Shewanella frigidimarina NCIMB400 grown anaerobically with ferric citrate or ferric pyrophosphate as the sole terminal electron acceptor, but not in anaerobic cultures of the bacterium with other respiratory substrates. Ifc(3) was purified to homogeneity and revealed by biochemical, spectroscopic and primary structure analyses to contain four low-spin bis-His-ligated c(3)-haems, with midpoint reduction potentials of -73, -141, -174 and -259 mV. A low-potential flavin was present in the form of non-covalently bound FAD; the protein possessed a unidirectional fumarate reductase activity. Disruption of the chromosomal gene encoding Ifc(3), ifcA, did not lead to a significant change in the rate of Fe(3+) reduction in batch culture. However, during such growth the Ifc(3)-deficient mutant produced both a 35 kDa periplasmic c-type cytochrome and a 45 kDa membrane-associated c-type cytochrome at markedly higher levels than did the parent strain. Nucleotide sequencing data from directly upstream of ifcA indicated the presence of an open reading frame encoding a putative outer-membrane beta-barrel protein of 324 amino acid residues.