Oxidation of the his-52 -> leu mutant of cytochrome c peroxidase by p-nitroperoxybenzoic acid: Role of the distal histidine in hydroperoxide activation

Both cytochrome c peroxidase (CcP) and a mutant cytochrome c peroxidase in which the distal histidine has been replaced by leucine, CcP(H52L), are con verted to hydroxy-ligated derivatives at alkaline pH, In CcP, the hydroxy-l igated derivative is subsequently converted to a bis-imidazole species prio r to protein denaturation while the initial hydroxy-ligated CcP(H52L) is co nverted to a second, spectroscopically distinct hydroxy-ligated species pri or to denaturation. The spectra of the alkaline forms of CcP and CcP(H52L) have been determined between 310 and 700 nm. The pH dependence of the rate of reaction between CcP(H52L) and hydrogen peroxide has been extended to pH 10. The hydroxy-ligated form of CcP(H52L) reacts with hydrogen peroxide 4 times more rapidly than the pentacoordinate, high-spin form of CcP(H52L) th at exists at neutral pH, The rate of the reaction between p-nitroperoxybenz oic acid and CcP(H52L) has been measured between pH 4 and pH 8. Neutral p-n itroperoxybenzoic acid reacts with CcP(H52L) 10(5) times more slowly than w ith CcP while the negatively charged p-nitroperoxybenzoate reacts with CcP( H52L) 10(3) times more slowly than with CcP, These data indicate that the r ole of the distal histidine during the initial formation of the peroxy anio n/heme iron complex is not simply base catalysis.