pH-Dependent spectral and kinetic properties of cytochrome c peroxidase: comparison of freshly isolated and stored enzyme

Vitello, Lidia B.; Huang, Min; Erman, James E.

doi:10.1021/bi00470a004

Cited by 71 publications

(96 citation statements)

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“…Thus, ascorbate peroxidase and cytochrome c peroxidase have comparable rates of reaction with hydrogen peroxide [17], but markedly different reactivities with the small reducing substrate, ascorbate.…”

Section: Discussionmentioning

confidence: 99%

Kinetic and spectral properties of pea cytosolic ascorbate peroxidase

et al. 1996

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Sufficient highly purified native pea cytosolic ascorbate peroxidase was obtained to characterize some of its kinetic and spectral properties. Its rate constant for compound I formation from reaction with Hz02 is 4.0X 10' M-' s-l, somewhat faster than is typical for peroxidases. Compound I has the typical optical spectrum of an iron(porphyrin-n-cation radical, despite considerable homology with yeast cytochrome c peroxidase. The rate constant for cornround I reduction by ascorbate is extremely fast (8.0 X 10' M-s ' at pH 7.8), again in marked contrast to the behavior of the yeast enzyme. The pHrate profde for compound I formation indicates a pK, value of 5.0 for a group affecting the active site reaction.

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Section: Discussionmentioning

confidence: 99%

Kinetic and spectral properties of pea cytosolic ascorbate peroxidase

et al. 1996

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“…5 in nitrate containing buffers [2,32,331) or the Arg48-Leu variant (5.8 in phosphate and phosphate/ nitrate buffers [71).…”

Section: Discussionmentioning

confidence: 99%

Charge Reversal of a Critical Active‐Site Residue of Cytochrome‐c Peroxidase

Bujons

Dikiy

Ferrer

et al. 1997

European Journal of Biochemistry

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A new variant of cytochrome-c peroxidase in which the positively charged Arg48 present in the distal heme-binding pocket has been replaced with a Glu residue has been prepared and characterized to explore, in part, the possibility that a negative charge close to the heme could contribute to stabilization of a porphyrin-centered n-cation radical in the compound I derivative of the variant. Between pH 4 and 8, this variant forms three pH-linked spectroscopic species. The electronic absorption and 'H-NMR spectra of the predominant form at low pH (HS1) are indicative of a high-spin, pentacoordinate heme iron system. Near neutral pH, a second high-spin species (HS2) is dominant, in which the heme iron center is hexacoordinated, with a water molecule as the sixth axial ligand. At high pH, the third form (LS) exhibits the spectroscopic characteristics of a low-spin, hexacoordinate heme center with bishistidine axial ligation. The apparent pK, values for these transitions are 4.4 and 7.4, respectively, in phosphate buffers and 5.0 and 7.1, respectively, in phosphatehitrate buffers. Replacement of Arg48 with Glu reduces the thermal stability of the enzyme and also decreases the Fe(III)/Fe(II) reduction potential of the enzyme by approximately 50 mV relative to that of the wild-type enzyme. The stability of compound I formed by the variant is decreased although the rate at which it forms is just one order of magnitude less than that of the wild-type enzyme, thus confirming previous results which indicate that the function of residue 48 in the wild-type peroxidase is more related to the stability of compound I than to its formation [Erman, J.

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“…Extinction coefficients and positions of the Soret maxima for the various proteins at pH 6.0 are: yCcP, 98 ± 3 mM −1 cm −1 at 408 nm; rCcP, 101 ± 3 mM −1 cm −1 at 408 nm; reduced cytochrome c(C102T), 150 ± 5 mM −1 cm −1 at 414 nm; and oxidized yeast cytochrome c(C102T), 118 ± 4 mM −1 cm −1 at 408 nm (29,39).…”

Section: Protein Concentration Determinationmentioning

confidence: 99%

“…b RZ is the purity number, the ratio of the absorbance at the Soret maximum relative to the absorbance at the maximum of the protein band near 280 nm. c Data from studies reported in references (39,44). c Data from studies reported in references (39,44).…”

Section: Supplementary Materialsmentioning

confidence: 99%

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Effect of Single-Site Charge-Reversal Mutations on the Catalytic Properties of Yeast Cytochrome c Peroxidase: Mutations near the High-Affinity Cytochrome c Binding Site

et al. 2007

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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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pH-Dependent spectral and kinetic properties of cytochrome c peroxidase: comparison of freshly isolated and stored enzyme

Cited by 71 publications

References 20 publications

Kinetic and spectral properties of pea cytosolic ascorbate peroxidase

Kinetic and spectral properties of pea cytosolic ascorbate peroxidase

Charge Reversal of a Critical Active‐Site Residue of Cytochrome‐c Peroxidase

Effect of Single-Site Charge-Reversal Mutations on the Catalytic Properties of Yeast Cytochrome c Peroxidase: Mutations near the High-Affinity Cytochrome c Binding Site

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