Relocation of the Distal Histidine in Cytochrome c Peroxidase: Properties of CcP(W51H), CcP(W51H/H52W), and CcP(W51H/H52L)

Abstract

Many heme proteins have distal histidine residues that play important roles in determining heme protein reactivity. These distal histidines are in significantly different orientations,and distances from the heme iron in different heme proteins and the position of the distal histidine relative to the heme iron can influence reactivity at the heme center. To explore the effect of distal histidine position on the properties of cytochrome c peroxidase (CcP), three CcP mutants in which tryptophan 51 was replaced with a histidine residue were constructed. All three mutants, CcP(W51H), CcP(W51H/H52W), and CcP(W51H/H52L), have altered electronic absorption spectra, indicating that the heme group in the mutants is six-coordinate rather than five-coordinate as it is in wild-type CcP. The hydrogen peroxide reaction rate is 56-6200-fold slower for the mutants than for wild-type CcP. All three mutants form a CcP Compound I-like intermediate, in which the Fe(IV) site decays between 500 and 3000 times more rapidly than the Fe(IV) site in wild-type CcP Compound I. The W51H mutations have a weaker effect on cyanide binding, with the cyanide affinity only 2-8 times weaker than for CcP. The cyanide association rate constants are between 5 and 85 times slower for the W51H mutants, while the cyanide dissociation rate constants range from 3 times slower to 6 times faster than those of wild-type CcP.