SITE-DIRECTED MUTAGENESIS OF A PHENYLALANINE RESIDUE STRICTLY CONSERVED IN CYTOCHROMES C(3)

Reduction of the haems in tetrahaem cytochromes C-3 is a cooperative p rocess, i.e., reduction of each of the haems depends on the redox stat es of the other haems. Furthermore, electron transfer is coupled to pr oton transfer (redox-Bohr effect). Two of its haems and a strictly con served nearby phenylalanine residue, F20, in Desulfovibrio vulgaris (H ildenborough) cytochrome c(3) form a structural motif that is present in all cytochromes c(3) and also in cytochrome c oxidase. A putative r ole for this phenylalanine residue in the cooperativity of haem reduct ion was investigated. Therefore, this phenylalanine was replaced, with genetic techniques, by isoleucine and tyrosine in D. vulgaris (Hilden borough) cytochrome c(3). Cyclic voltammetry studies revealed a small increase (30 mV) in one of the macroscopic redox potentials in the mut ated cytochromes. EPR showed that the main alterations occurred in the Vicinity of haem I, the haem closest to residue 20 and one of the hae ms responsible for positive cooperativities in electron transfer of D. vulgaris cytochrome c(3). NMR studies of F20I cytochrome c(3) demonst rated that the haem core architecture is maintained and that the more affected haem proton groups are those near the mutation site. NMR redo x titrations of this mutated protein gave evidence for only small chan ges in the relative redox potentials of the haems. However, electron/e lectron and proton/electron cooperativity are maintained, indicating t hat this aromatic residue has no essential role in these processes. Fu rthermore, chemical modification of the N-terminal amino group of cyto chrome C-3 backbone, which is also very close to haem I, had no effect on the network of cooperativities.