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  • 1
    ISSN: 1432-1327
    Keywords: Key words Nine-haem cytochrome c ; Multiple wavelength anomalous dispersion phasing method ; Sulfate-reducing bacteria ; Electron transfer ; Modelling and interaction studies
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Abstract  A monomeric nine-haem cytochrome c (9Hcc) with 292 amino acid residues was isolated from cells of the sulfate- and nitrate-reducing bacterium Desulfovibrio desulfuricans ATCC 27774 grown under both nitrate- and sulfate-respiring conditions. The nucleotide sequence encoding the 292 residues was determined, allowing the correction of about 10% of the previous primary structure, determined from 1.8 Å electron density maps. The refinement at 1.8 Å resolution of the structural model was completed, giving an R-value of 16.5%. The nine haem groups are arranged into two tetrahaem clusters, located at both ends of the molecule, with Fe-Fe distances and local protein fold very similar to tetrahaem cytochromes c 3, and the extra haem is located asymmetrically between the two regions. The new primary sequence determination confirmed the 39% sequence homology found between this cytochrome and the C-terminal region (residues 229–514) of the high-molecular-weight cytochrome c (Hmc) from D. vulgaris Hildenborough, providing strong evidence of structural similarity between 9Hcc and the C-terminal region of Hmc. The interaction between 9Hcc and the tetrahaem cytochrome c 3 from the same organism was studied by modelling methods, and the results suggest that a specific interaction is possible between haem 4 of tetrahaem cytochrome c 3 and haem 1 or haem 2 of 9Hcc, in agreement with previous kinetic experiments which showed the catalytic effect of the tetrahaem cytochrome c 3 upon the reduction of 9Hcc by the [NiFe] hydrogenase from D. desulfuricans ATCC 27774. These studies suggest a role for 9Hcc as part of the assembly of redox proteins involved in recycling the molecular hydrogen released by the cell as a result of substrate oxidation.
    Type of Medium: Electronic Resource
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  • 2
    ISSN: 1432-1327
    Keywords: Key words Electron-proton transfer coupling ; Redox-Bohr effect ; Electrostatic calculations ; pKa calculations ; Cytochrome c3
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Abstract  The pH dependence of the redox potentials in the tetrahemic cytochrome c 3 from Desulfovibrio vulgaris Hildenborough (redox-Bohr effect) is here investigated using continuum electrostatics methods. The redox-Bohr effect seems to be associated with changes in the protonation state of charged residues in the protein, but the exact residues had not been identified. The global pK a of this phenomenon is dependent on the redox state of the molecule, and the influence of the pH on the microscopic potential of each heme has been experimentally quantified. The availability of detailed experimental data provides us with important and unique guides to the performance of ab initio pK a calculations aiming at the identification of the groups involved. These calculations were performed in several redox states along the reduction pathway, with the double objective of finding groups with redox-linked pK a shifts, and absolute pK as compatible with the redox-Bohr effect. The group with the largest pK a shift along the reduction pathway is propionate D from heme I. Its effect on the redox potential of individual hemes, as calculated by electrostatic calculations, correlates very well with the experimental order of influence, making it a likely candidate. Abnormal titration of the same propionate has been experimentally observed on a homologous cytochrome c 3 from a different strain, thus strengthening the theoretical result. However, its absolute calculated pK a in the fully oxidised cytochrome is outside the zone where the phenomenon is known to occur, but the calculation shows a strong dependence on small conformational changes, suggesting large uncertainties in the calculated value. A group with a pK a value within the experimentally observed range is propionate D from heme IV. Its influence on the redox potential of the hemes does not correlate with the experimental order, indicating that, although it may be one of the possible players on the phenomenon, it cannot be solely responsible for it. Mutation of the Lys45 residue is suggested as an indirect way of probing the importance of the propionate D from heme I in the mechanism. Non-heme groups may also be involved in this process; our calculations indicate His67 and the N-terminal as groups that may play a role. Accuracy and applicability of current continuum electrostatic methods are discussed in the context of this system.
    Type of Medium: Electronic Resource
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  • 3
    ISSN: 1432-1327
    Keywords: Flavodoxin Cytochrome Electron transfer Electrostatic Stopped-flow kinetics
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Abstract. A hypothetical model for the non-physiological electron transfer complex between cytochrome c 553 (c 553) and the flavodoxin (fld) from the sulphate-reducing bacteria Desulfovibrio vulgaris has been recently published [1] based on rigid-body docking and refined by molecular dynamics. In this study, the functional validity of this model is tested by looking at the role of electrostatics in the non-physiological inter-protein electron transfer between the two proteins at different ionic strengths. The results are compared with the electron transfer between fld and cytochrome c from horse heart (hhc). Second-order rate constants (k2) were measured for both non-physiological systems at different ionic strengths: a complex, bell-shaped behaviour is observed for the k2 of the c 553/fld redox pair with an optimum rate at I=58 mmol l–1, whereas under the same conditions the k2 for hhc/fld decreased monotonically with increasing ionic strength. Results from the electron transfer kinetics are rationalised in terms of reorganisational effects of an ensemble of conformations of the electron transfer competent c 553/fld complexes, consistent with the published model.
    Type of Medium: Electronic Resource
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  • 4
    ISSN: 1432-1327
    Keywords: Key words Protein electrostatistics ; Electron-proton coupling ; Redox-Bohr effect ; pKa calculations ; Cytochrome c3
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Abstract  A comparative study of the pH-dependent redox mechanisms of several members of the cytochrome c 3 family has been carried out. In a previous work, the molecular determinants of this dependency (the so-called redox-Bohr effect) were investigated for one species using continuum electrostatic methods to find groups with a titrating range and strength of interaction compatible with a mediating role in the redox-Bohr effect. Here we clarify these aspects in the light of new and improved pK a calculations, our findings supporting the hypothesis of propionate D from heme I being the main effector in the pH-dependent modulation of the cytochrome c 3 redox potentials in all the c 3 molecules studied here. However, the weaker (but significant) role of other titrating groups cannot be excluded, their importance and identity changing with the particular molecule under study. We also calculate the relative redox potentials of the four heme centers among the selected members of the c 3 family, using a continuum electrostatic method that takes into account both solvation and interaction effects. Comparison of the calculated values with available data for the microscopic redox potentials was undertaken, the quality of the agreement being dependent upon the choice of the dielectric constant for the protein interior. We find that high dielectric constants give best correlations, while low values result in better magnitudes for the calculated potentials. The possibility that the crystallographic calcium ion in c 3 from Desulfovibrio gigas may be present in the solution structure was tested, and found to be likely.
    Type of Medium: Electronic Resource
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  • 5
    ISSN: 1432-1327
    Keywords: Key words Electron transfer ; Protein-protein interactions ; Ionic strength effects in electron transfer ; Docking calculations ; Electron transfer calculations
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Abstract  Theoretical studies of protein-protein association and electron transfer were performed on the binary systems formed by Desulfovibrio vulgaris Hildenborough (D. v. H.) flavodoxin and D. v. H. cytochrome c 553 and by flavodoxin and horse heart cytochrome c. Initial structures for the complexes were obtained by rigid-body docking and were refined by MD to allow for molecular flexibility. The structures thus obtained were analysed in terms of their relative stability through the calculation of excess energies. Electrostatic, van der Waals and solvation energy terms showed all to have significant contributions to the stability of complexes. In the best association solutions found for both cytochromes, these bind to different zones of flavodoxin. The binding site of flavodoxin observed for cytochrome c is in accordance with earlier works [27]. The various association modes found were characterised in terms of electron transfer using the Pathways model. For complexes between flavodoxin and horse heart cytochrome c, some correlation was observed between electron tunnelling coupling factors and conformation energy; the best conformation found for electron transfer corresponded also to the best one in terms of energy. For complexes between flavodoxin and cytochrome c 553 this was not the case and a lower correlation was observed between electron tunnelling coupling factors and excess energies. These results are in accordance with the differences in the experimental dependence of electron transfer rates with ionic strength observed between these two cases.
    Type of Medium: Electronic Resource
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