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

摘要

Many heme proteins have distal histidine residues that play important roles in determining hemenprotein reactivity. These distal histidines are in significantly different orientations,and distances from thenheme iron in different heme proteins and the position of the distal histidine relative to the heme iron canninfluence reactivity at the heme center. To explore the effect of distal histidine position on the properties ofncytochrome c peroxidase (CcP), three CcP mutants in which tryptophan 51 was replaced with a histidinenresidue were constructed. All three mutants, CcP(W51H), CcP(W51H/H52W), and CcP(W51H/H52L), havenaltered electronic absorption spectra, indicating that the heme group in the mutants is six-coordinate rathernthan five-coordinate as it is in wild-type CcP. The hydrogen peroxide reaction rate is 56-6200-fold slower fornthe mutants than for wild-type CcP. All three mutants form a CcP Compound I-like intermediate, in whichnthe Fe(IV) site decays between 500 and 3000 times more rapidly than the Fe(IV) site in wild-type CcPnCompound I. The W51H mutations have a weaker effect on cyanide binding, with the cyanide affinity onlyn2-8 times weaker than for CcP. The cyanide association rate constants are between 5 and 85 times slower fornthe W51H mutants, while the cyanide dissociation rate constants range from 3 times slower to 6 times fasternthan those of wild-type CcP