Desulfovibrio vulgaris (Hildenborough) cytochrome c(3) probed by site-specific mutagenesis

Cytochromes c(3) isolated from Desulfovibrio spp. are periplasmic proteins that play a central role in energy transduction by coupling the transfer of electrons and protons from hydrogenase. Comparison between the oxidized an d reduced structures of cytochrome c(3) isolated from Desulfovibrio vulgari s (Hildenborough) show that the residue threonine 24, located in the vicini ty of heme III, reorients between these two states [Messias, A. C., Kastrau , D. H. W., Costa, H. S., LeGall, J., Turner, D. L., Santos, H., and Xavier , A. V. (1998) J. Mol. Biol. 281, 719-739]. Threonine 24 was replaced with valine by site-directed mutagenesis to elucidate its effect on the redox pr operties of the protein. The NMR spectra of the mutated protein are very si milar to those of the wild type, showing that the general folding and heme core architecture are not affected by the mutation. However, thermodynamic analysis of the mutated cytochrome reveals a large alteration in the micros copic reduction potential of heme III (75 and 106 mV for the protonated for ms of the fully reduced and oxidized states, respectively). The redox inter actions involving this heme are also modified, while the remaining heme-hem e interactions and the redox-Bohr interactions are less strongly affected. Hence, the order of oxidation of the hemes in the mutated cytochrome is dif ferent from that in the wild type, and it has a higher overall affinity for electrons. This is consistent with the replacement of threonine 24 by vali ne preventing the formation of a network of hydrogen bonds, which stabilize s the oxidized state. The mutated protein is unable to perform a concerted two-electron step between the intermediate oxidation stages, 1 and 3, which can occur in the wild-type protein. Thus, replacing a single residue unbal ances the global network of cooperativities tuned to control thermodynamica lly the directionality of the stepwise electron transfer and may affect the functionality of the protein.