Reduction Potential Shifts Due to Active Site and Surface Mutations of Yeast Cytochrome c Peroxidase

Abstract

Twenty two reduction potentials of yeast cytochrome c peroxidase (CcP) mutants were determined at pH 7.0 in order to determine the effect of both surface and heme pocket mutations on the Fe(III)/Fe(II) redox couple of CcP, as well as to determine redox potential range that could be obtained through enzyme point mutations. The mutations include variants in the distal and proximal heme pockets as well as on the enzyme surface and involve single, double, and triple point mutations. Compared to the reduction potential of the wild- type enzeme, both negative and positive shifts in the reduction potential were observed, spanning a range of 113 mV. The His-52→Asn mutation gave the most negative potential, -259 mV, while a triple mutant in which the three distal pocket residues, Arg-48, Trp-51, and His-52, were all converted to leucine residues gave the most positive potential, -146 mV.