A. Dolla et al., CHARACTERIZATION AND OXIDOREDUCTION PROPERTIES OF CYTOCHROME-C(3) AFTER HEME AXIAL LIGAND REPLACEMENTS, The Journal of biological chemistry, 269(9), 1994, pp. 6340-6346
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.