The crystal structure and reactivity with hydrogen peroxide are reported for a mutant of a cloned cytochrome c peroxidase [CcP(MI)], in which the conserved distal His (His-52) is replaced with Leu. The reaction of the H52L enzyme with peroxide was examined as a function of pH in 0.1 M phosphate buffers and buffers in which nitrate was used to adjust the ionic strength. The pH-independent bimolecular rate constant for the reaction of H52L with peroxide was 731 +/- 44 and 236 +/- 14 M-1 s-1 in phosphate and nitrate-containing buffers, respectively. This represents a 10(5)-fold decrease in rate relative to the CcP(MI) parent under comparable conditions. Single-crystal diffraction studies showed that no dramatic changes in the structure or in the accessibility of the heme binding site were caused by the mutation. Rather, the mutation caused significant structural changes only at residue 52 and the nearby active-site water molecules. The residual reactivity of the H52L enzyme with peroxide was pH- and buffer-dependent. In nitrate-containing buffer, the apparent bimolecular rate constant for the reaction with peroxide decreased with decreasing pH; the loss of reactivity correlated with protonation of a group with an apparent pKA = 4.5. Protonation of the group caused a loss of reactivity with peroxide. This is in contrast to the CcP(MI) parent enzyme, as well as all other mutants that have been examined, where the loss of reactivity correlates with protonation of an enzyme group with an apparent pKA = 5.4.(ABSTRACT TRUNCATED AT 250 WORDS)

The crystallographic structures of two cytochrome c peroxidase (CcP) mutants, CcP(R48L) and CcP(R48K), have been determined. In addition, the electronic absorption spectrum and the hydrogen peroxide reactivity of these two mutants have been determined between pH 4 and 8. Both the crystallographic structure and the electronic absorption spectrum of CcP(R48L) are consistent with exclusive pentacoordination of the heme iron between pH 4 and 6.5. At higher pH, CcP(R48L) forms an alkaline bis-imidazole form of CcP with the distal histidine coordinated to the heme iron. The apparent pKA for this transition is 7.5 in CcP(R48L). The observed pseudo-first-order rate constant for the reaction between CcP(R48L) and hydrogen peroxide saturates at high peroxide concentrations. The data are consistent with a rate-limiting oxygen-oxygen bond scission at high peroxide concentrations. The observed rate of the bond scission step ranges between 1000 and 1950 s-1, an estimated 2 orders of magnitude slower than for wild-type enzyme. The data suggest that the protonated form of His-52 increases the bond scission step by a factor of 2. The properties of the CcP(R48K) mutant are significantly different from those of CcP(R48L). The crystal structure of CcP(R48K) shows Lys-48 occupying the putative peroxide binding site. The electronic absorption spectrum indicates that CcP(R48K) is predominantly pentacoordinate at neutral pH but with detectable amounts of hexacoordinate forms. Two ionizable groups affect the electronic absorption spectrum of CcP(R48K). An apparent ionization near pH 4 produces an enzyme with increased hexacoordination, while an apparent pKA of 6.9 generates the alkaline bis-imidazole form. The peroxide reaction saturates at high peroxide concentrations for CcP(R48K) and is attributed to a conformational-gating mechanism. The maximum rate for the reaction between CcP(R48K) and hydrogen peroxide is probably limited by the movement of either Lys-48 or His-52. This rate is 200 and 290 s-1 in nitrate-containing buffers and phosphate buffers, respectively. Evidence is provided that Arg-48 in wild-type enzyme is responsible for nitrate binding in the heme pocket and for stabilizing CcP Compound I.

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The effect of long-term storage on the electronic absorption spectrum and the kinetic properties of cytochrome c peroxidase has been investigated. No detectable differences were observed between freshly isolated enzyme and enzyme stored below -20 degrees C, in the crystalline state, for up to 41 months. The electronic absorption spectrum and the rate of the enzyme-hydrogen peroxide reaction are essentially independent of pH in 0.1 M potassium phosphate buffers for both fresh and stored enzyme. In buffers containing KNO3, the absorption spectrum and the kinetic properties of both fresh and stored enzyme vary with pH, consistent with the titration of an ionizable group with an apparent pKa of 5.5 +/- 0.1. The differences between phosphate- and nitrate-containing buffers are attributed to specific ion effects. In KNO3-containing buffers, the high-pH form of the enzyme reacts rapidly with hydrogen peroxide while the low-pH form is unreactive. Evidence is presented which indicates that both the low-pH and high-pH forms of the enzyme in KNO3-containing buffers are 5-coordinate, high-spin Fe(III) species.

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