Forty-six charge-reversal mutants of yeast cytochrome c peroxidase (CcP) have been constructed in order to determine the effect of localized charge on the catalytic properties of the enzyme. The mutants include the conversion of all 20 glutamate residues and 24 of the 25 aspartate residues in CcP, one at a time, to lysine residues. In addition, two positive-to-negative charge-reversal mutants, R31E and K149D, are included in the study. The mutants have been characterized by absorption spectroscopy and hydrogen peroxide reactivity at pH 6.0 and 7.5 and by steady-state kinetic studies using recombinant yeast iso-1 ferrocytochrome c (C102T) as substrate at pH 7.5. Many of the charge-reversal mutations cause detectable changes in the absorption spectrum of the enzyme reflecting increased amounts of hexacoordinate heme compared to wild-type CcP. The increase in hexacoordinate heme in the mutant enzymes correlates with an increase in H 2O 2-inactive enzyme. The maximum velocity of the mutants decreases with increasing hexacoordination of the heme group. Steady-state velocity studies indicate that 5 of the 46 mutations (R31E, D34K, D37K, E118K, and E290K) cause large increases in the Michaelis constant indicating a reduced affinity for cytochrome c. Four of the mutations occur within the cytochrome c binding site identified in the crystal structure of the 1:1 complex of yeast cytochrome c and CcP [Pelletier, H., and Kraut, J. (1992) Science 258, 1748-1755] while the fifth mutation site lies outside, but near, the crystallographic site. These data support the hypothesis that the CcP has a single, catalytically active cytochrome c binding domain, that observed in the crystal structures of the cytochrome c/CcP complex.

Fifteen single-site charge-reversal mutations of yeast cytochrome c peroxidase (CcP) have been constructed in order to determine the effect of localized charge on the catalytic properties of the enzyme. The mutations are located on the front face of CcP, near the cytochrome c binding site identified in the crystallographic structure of the yeast cytochrome c/CcP complex (Pelletier and Kraut (1992) Science 258, 1748-1755. The mutants are characterized by absorption spectroscopy and hydrogen peroxide reactivity at both pH 6.0 and 7.5 and by steady-state kinetic studies using recombinant yeast iso-1 ferrocytochrome c(C102T) as substrate at pH 7.5. Some of the chargereversal mutations cause detectable changes in the absorption spectrum, especially at pH 7.5, reflecting changes in the equilibrium between penta-and hexa-coordinate heme species in the enzyme. An increase in the amount of hexa-coordinate heme in the mutant enzymes correlates with an increase in the fraction of enzyme that does not react with hydrogen peroxide. Steady-state velocity measurements indicate that five of the fifteen mutations cause large increases in the Michaelis constant (R31E, D34K, D37K, E118K, and E290K). These data support the hypothesis that the cytochrome c/CcP complex observed in the crystal is the dominant catalytically active complex in solution.Cytochrome c peroxidase, CcP 1 , is a detoxification enzyme localized between the inner and outer membranes of yeast mitochondria (1). CcP decreases toxic levels of hydrogen peroxide by catalyzing its reduction to water using ferrocytochrome c (2). The catalytic mechanism involves oxidation of the native enzyme by hydrogen peroxide to an enzyme intermediate called CcP Compound I, CcP-I. CcP-I contains two oxidized sites, an oxyferryl Fe(IV) heme group and a tryptophan π-cation radical located within van der Waals distance of the heme. Interaction with, and electron transfer from, ferrocytochrome c reduces CcP-I back to the native state via a second enzyme intermediate, CcP Compound II, CcP-II, completing the catalytic cycle. Since 1980, when the three-dimensional structure of CcP was first reported (3,4), CcP has played an important role in elucidating the structural basis for heme protein reactivity, † This work was supported in part by a grant from the National Institutes of Health (R15 GM59740). * Corresponding author. Phone: (815) . E-mail: jerman@niu.edu. 1 Abbreviations: Mutations in the amino acid sequences of either CcP or cytochrome c are indicated by using the one letter code for the amino acid residue in the wild-type protein, followed by the residue number and the one letter code for the amino acid residue in the mutant protein, i.e., C102T represents a mutant in which a threonine residue replaces the cysteine residue at position 102 of the wildtype protein; CcP, generic abbreviation for cytochrome c peroxidase whatever the source; yCcP, authentic yeast cytochrome c peroxidase isolated from bakers' yeast, Saccharomyces cervisiae; rCcP, recombinant CcP expressed in E. ...

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Previously, we constructed, expressed, and purified 46 charge-reversal mutants of yeast cytochrome c peroxidase (CcP) and determined their electronic absorption spectra, their reaction with H2O2, and their steady-state catalytic properties [ Pearl , N. M. et al. (2008) Biochemistry 47 , 2766 - 2775 ]. Forty-four of the mutants involve the conversion of either an aspartate or glutamate residue to a lysine residue, while two are positive-to-negative mutations, R31E and K149D. In this paper, we report on a calorimetric study of the interaction of each charge-reversal mutant (excluding the internal mutants D76K and D235K) with recombinant yeast iso-1 ferricytochrome c(C102T) (yCc) under conditions where only one-to-one yCc/CcP complex formation is observed. Thirteen of the 44 surface-site charge-reversal mutants decrease the binding affinity for yCc by a factor of 2 or more. Eight of the 13 mutations (E32K, D33K, D34K, E35K, E118K, E201K, E290K, E291K) occur within, or on the immediate periphery, of the crystallographically defined yCc binding site [ Pelletier , H. and Kraut , J. (1992) Science 258 , 1748 - 1755 ], three of the mutations (D37K, E98K, E209K) are slightly removed from the crystallographic site, and two of the mutations (D165K, D241K) occur on the "back-side" of CcP. The current study is consistent with a model for yCc binding to CcP in which yCc binds predominantly near the region defined by crystallographic structure of the 1:1 yCc-CcP complex, whether as a stable electron-transfer active complex or as part of a dynamic encounter complex.