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.