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Focuses on the use of the site-directed mutagenesis of charged residues in the vicinity of heme I, to investigate the structural basis for the pH dependence of the redox potential in the tetrahemic Desulfovibrio vulgaris cytochrome. Performance of the mutation of lysine 45, located in the neighborhood of the propionates of heme I; Indication of electron paramagnetic resonance (EPR) and nuclear magnetic resonance (NMR) data.

A c-type monohemic ferricytochrome c552 (11 kDa) was isolated from the soluble extract of a marine denitrifier. Pseudomonas nautica strain 671, grown under anaerobic conditions with nitrate as final electron acceptor. The NH2-terminal sequence and the amino acid composition of the cytochrome were determined. The heme iron of the cytochrome c552 has histidine-methionine as axial ligands, and a pH-dependent mid-point redox potential, equal to 250 mV at pH 7.6. The presence of methionine was demonstrated by visible. EPR and NMR spectroscopies. The assignment of most of the hemic protons was performed applying two-dimensional NOE spectroscopy (NOESY), and the aromatic region was assigned through two-dimensional correlated spectroscopy (COSY) experiments. The EPR spectrum of the oxidised form of the cytochrome c552 is typical of a low-spin ferric heme. [ABSTRACT FROM AUTHOR]

A c-type monoheme ferricytochrome c550 (9.6 kDa) was isolated from cells of Bacillus halodenitrificans sp.nov., grown anaerobically as a denitrifier. The visible absorption spectrum indicates the presence of a band at 695 nm characteristic of heme—methionine coordination. The midpoint redox potential was determined at several pH values by visible spectroscopy. The redox potential at pH 7.6 is 138 mV. When studied by ¹H-NMR spectroscopy as a function of pH, the spectrum shows a pH dependence with pKa values of 6.0 and 11.0. According to these pKa values, three forms designated as I, II and Ill can be attributed to cytochrome c550. The first pKa is probably associated with protonation of the propionate groups. The second pKa value introduces a larger effect in the ¹H-NMR spectrum and is probably due to the ionisation of the axial histidine. Studies of temperature variation of the ¹H-NM R spectra for both the ferrous and ferri forms of the cytochrome were performed. Heme meso protons, the heme methyl groups, the thioether protons, two protons from a propionate and the methylene protons from the axial methionine were identified in the reduced form. The heine methyl resonances of the ferri form were also assigned. EPR spectroscopy was also used to probe the ferric heine environment. A signal at gmax ≈ 3.5 at pH 7.5 was observed indicating an almost axial heine environment. At higher pH values the signal at gmax ≈ 3.5 converts mainly to a signal at g ≈ 2.96. The pKa associated with this change is around 11.3. The N-terminal sequence of this cytochrome was determined and compared with known amino acid sequences of other cytochromes. [ABSTRACT FROM AUTHOR]

A c-type monoheme cytochrome c554 (13 kDa) was isolated from cells of Achromobacter cycloclastes IAM 1013 grown anaerobically as a denitrifier. The visible absorption spectrum indicates the presence of a band at 695 nm characteristic of heme-methionine coordination (low-spin form) coexisting with a minor high-spin form as revealed by the contribution at 630 nm. Magnetic susceptibility measurements support the existence of a small contribution of a high-spin form at all pH values, attaining a minimum at intermediate pH values. The mid-point redox potential determined by visible spectroscopy at pH 7.2 is + 150 mV. The pH-dependent spin equilibrum and other relevant structural features were studied by 300-MHz m 1H-NMR spectroscopy. In the oxidized form, the 1H-NMR spectrum shows pH dependence with pKa values at 5.0 and 8.9. According to these pKa values, three forms designated as I, II and III can be attributed to cytochrome c554. Forms I and II predominate at low pH values, and the 1H-NMR spectra reveal heine methyl proton resonances between 40 ppm and 22 ppm. These forms have a methionyl residue as a sixth ligand, and C6 methyl group of the bound methionine was identified in the low-field region of the NMR spectra. Above pH 9.6, form III predominates and the 1H-NMR spectrum is characterized by down-field hyperfine-shifted heine methyl proton resonances between 29 ppm and 22 ppm. Two new resonances are observed at [This symbol cannot be presented in ASCII format]66 ppm and 54 ppm, and are taken as indicative of a new type of heine coordination (probably a lysine residue). These pH-dependent features of the 1H-NMR spectra are discussed in terms of the heine environment structure. The chemical shifts of the methyl resonances at different pH values exhibit anti-Curie temperature dependence. In the ferrous state, the 1H-NMR spectrum shows a methyl proton resonance at -3.9 ppm characteristic of methionine axial ligation. The electron-transfer rate between ferric and ferrous forms has been estimated to be smaller than 2 × l04 M-1s-1 at pH 5. EPR spectroscopy was also used to probe the ferric heine environment. A prominent signal at gmax[This symbol cannot be presented in ASCII format]3.58 and the overall lineshape of the spectrum indicate an almost axial heme environment. [ABSTRACT FROM AUTHOR]

Some bacteria and archaea synthesize haem by an alternative pathway, which involves the sequestration of sirohaem as a metabolic intermediate rather than as a prosthetic group. Along this pathway the two acetic acid side-chains attached to C12 and C18 are decarboxylated by sirohaem decarboxylase, a heterodimeric enzyme composed of AhbA and AhbB, to give didecarboxysirohaem. Further modifications catalysed by two related radical SAM enzymes, AhbC and AhbD, transform didecarboxysirohaem into Fe-coproporphyrin III and haem respectively. The characterization of sirohaem decarboxylase is reported in molecular detail. Recombinant versions of D esulfovibrio desulfuricans, D esulfovibrio vulgaris and M ethanosarcina barkeri AhbA/ B have been produced and their physical properties compared. The D . vulgaris and M . barkeri enzyme complexes both copurify with haem, whose redox state influences the activity of the latter. The kinetic parameters of the D . desulfuricans enzyme have been determined, the enzyme crystallized and its structure has been elucidated. The topology of the enzyme reveals that it shares a structural similarity to the AsnC/ Lrp family of transcription factors. The active site is formed in the cavity between the two subunits and a AhbA/ B-product complex with didecarboxysirohaem has been obtained. A mechanism for the decarboxylation of the kinetically stable carboxyl groups is proposed. [ABSTRACT FROM AUTHOR]

Desulfovibrio species are Gram-negative anaerobic sulfate-reducing bacteria that colonize the human gut. Recently, Desulfovibrio spp. have been implicated in gastrointestinal diseases and shown to stimulate the epithelial immune response, leading to increased production of inflammatory cytokines by macrophages. Activated macrophages are key cells of the immune system that impose nitrosative stress during phagocytosis. Hence, we have analyzed the in vitro and in vivo responses of Desulfovibrio vulgaris Hildenborough to nitric oxide (NO) and the role of the hybrid cluster proteins (HCP1 and HCP2) and rubredoxin oxygen oxi-doreductases (ROOl and ROO2) in NO protection. Among the four genes, hcp2 was the gene most highly induced by NO, and the hcp2 transposon mutant exhibited the lowest viability under conditions of NO stress. Studies in murine macrophages revealed that D. vulgaris survives incubation with these phagocytes and triggers NO production at levels similar to those stimulated by the cytokine gamma interferon (IFN-γ). Furthermore, D. vulgaris hep and roo mutants exhibited reduced viability when incubated with macrophages, revealing that these gene products contribute to the survival of D. vulgaris during macrophage infection. [ABSTRACT FROM AUTHOR]

Hybrid cluster proteins (HCP) contain two types of Fe/S clusters, namely a [4Fe-4S]2+/1+ or [2Fe-2S]2+/1+ cluster and a novel type of hybrid cluster, [4Fe-2S-2O], in the as-isolated state. Although first isolated from anaerobic sulfate-reducing bacteria, the analysis of the genomic sequences reveals that genes encoding putative hybrid cluster proteins are present in a wide range of organisms, aerobic, anaerobic, or facultative, from the Bacteria, Archaea, and Eukarya domains. Despite a detailed spectroscopic and structural characterization, the precise physiological function of these proteins remained unknown. The present work shows that the transcription of the Escherichia coli hcp gene is induced by hydrogen peroxide, and this induction is regulated by the redox-sensitive transcriptional activator, OxyR. The E. coli hcp mutant strain exhibits higher sensitivity to hydrogen peroxide, a behavior that reverts to the wild type phenotype once a plasmid carrying the hcp gene is reintroduced. Furthermore, the purified HCPs from E. coli and Desulfovibrio desulfuricans ATCC 27774 show an alternative enzymatic activity, which under physiological conditions exhibited Km values for hydrogen peroxide (~0.3 mM) within the range of other peroxidases. Altogether, the results reveal that HCP is involved in oxidative stress protection. [ABSTRACT FROM AUTHOR]

Examines the role of hydrogen-bond networks in controlling reduction potentials of Desulfovibrio vulgaris cytochrome c[sub 3]. Comparison between the oxidized and reduced structure of cytochrome c; Alteration in the microscopic reduction potential of heme III.