THEORETICAL-STUDIES ON THE REDOX-BOHR EFFECT IN CYTOCHROME C(3) FROM DESULFOVIBRIO-VULGARIS HILDENBOROUGH

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-B ohr effect seems to be associated with changes in the protonation stat e of charged residues in the protein, but the exact residues had not b een identified. The global pK(a) of this phenomenon is dependent on th e redox state of the molecule, and the influence of the pH on the micr oscopic 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 nb initio pK(a) calculations aiming at the identification of the groups involved. These calculation s were performed in several redox states along the reduction pathway, with the double objective of finding groups with redox-linked pK(a) sh ifts, and absolute pK(a)s compatible with the redox-Bohr effect. The g roup with the largest pK(a) shift along the reduction pathway is propi onate D from heme I. Its effect on the redox potential of individual h emes, as calculated by electrostatic calculations, correlates very wel l with the experimental order of influence, making it a likely candida te. 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 calculate d pK(a) in the fully oxidised cytochrome is outside the zone where the phenomenon is known to occur, but the calculation shows a strong depe ndence on small conformational changes, suggesting large uncertainties in the calculated value. A group with a pK(a) value within the experi mentally observed range is propionate D from heme IV. Its influence on the redox potential of the hemes does not correlate with the experime ntal order, indicating that, although it may be one of the possible pl ayers on the phenomenon, it cannot be solely responsible for it. Mutat ion of the Lys45 residue is suggested as an indirect way of probing th e importance of the propionate D from heme I in the mechanism. Non-hem e groups may also be involved in this process; our calculations indica te His67 and the N-terminal as groups that may play a role. Accuracy a nd applicability of current continuum electrostatic methods are discus sed in the context of this system.