The pH dependence of the redox potentials in the tetrahemic cytochrome c3 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 heine 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(a)s compatible with the redox-Bohr effect. The group with the largest pK(a) shift along the reduction pathway is propionate D from heine 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 c3 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 he 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.
- Cytochrome c
- Electron-proton transfer coupling
- Electrostatic calculations
- pK(a) calculations
- Redox-Bohr effect