Site-directed mutagenesis of yeast cytochrome c peroxidase shows histidine 181 is not required for oxidation of ferrocytochrome c.

The long-distance electron transfer observed in the complex formed between ferrocytochrome c and compound I, the peroxide-oxidized form of cytochrome c peroxidase (CCP), has been proposed to occur through the participation of His 181 of CCP and Phe 87 of yeast iso-1 cytochrome c [Poulos, T. L., & Kraut, J. (1980) J. Biol. Chem. 255, 10322-10330]. We have examined the role of His 181 of CCP in this process through characterization of a mutant CCP in which His 181 has been replaced by glycine through site-directed mutagenesis. Data from single-crystal X-ray diffraction studies, as well as the visible spectra of the mutant CCP and its 2-equiv oxidation product, compound I, show that at pH 6.0 the protein is not dramatically altered by the His 181----Gly mutation. The rate of peroxide-dependent oxidation of ferrocytochrome c by the mutant CCP is reduced only 2-fold relative to that of the parental CCP, under steady-state conditions. Transient kinetic measurements of the intracomplex electron transfer rate from ferrous cytochrome c to compound I indicate that the rate of electron transfer within the transiently formed complex at high ionic strength (mu = 114 mM, pH = 6) is also reduced by approximately 2-fold in the mutant CCP protein. The relatively minor effect of the loss of the imidazole side chain at position 181 on the kinetics of electron transfer in the CCP-cytochrome c complex precludes an obligatory participation of His 181 in electron transfer from ferrous cytochrome c to compound I.(ABSTRACT TRUNCATED AT 250 WORDS)