CHARACTERIZATION AND OXIDOREDUCTION PROPERTIES OF CYTOCHROME-C(3) AFTER HEME AXIAL LIGAND REPLACEMENTS

Cytochrome c(3) (M(r) 13,000) is a tetrahemic cytochrome in which the four heme iron atoms are coordinated by 2 histidine residues at the ax ial positions. The presence of several oxidoreduction centers in the s ame molecule raises the question of their coupling. To investigate thi s mechanism, four single mutations were introduced in cytochrome c(3) by site-directed mutagenesis, leading to the replacement of each histi dine, the sixth axial ligand of the heme iron atom, by a methionine re sidue. Characterization of the new set of molecules using biochemical and biophysical techniques was carried out. The novel methionine was c orrectly coordinated to the iron atom of hemes 3 and 4 in H25M and H70 M cytochromes c(3), respectively, and this coordination induced a larg e increase in the oxidoreduction potential of the mutated heme. In con trast, in the case of H22M and H35M cytochromes c(3), in which the cor responding methionine is in an oxidized form, only slight changes in r edox potential values were observed. In H22M, H25M, and H35M cytochrom es c(3), two conformations of the molecule were possible, in which the methionine is either free or coordinated to the iron atom. The rate c onstants for the electron exchange reactions between the cytochrome mu tants and the hydrogenase were measured using electrochemical techniqu es. Distinct behaviors were revealed depending on the mutation. The va lues of the rate constants for the electron exchange reactions are int erpreted in terms of intramolecular electron exchange among the four h emes of the cytochrome.