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Reduction potential of yeast cytochrome c peroxidase and three distal histidine mutants: dependence on pH.

ABSTRACT: The pH dependence of the Fe(III) reduction potential, E(0)', for yeast cytochrome c peroxidase (yCcP) and three distal pocket mutants, CcP(H52L), CcP(H52Q), and CcP(R48L/W51L/H52L), has been determined between pH 4 and 8. E(0)' values at pH 7.0 for the yCcP, CcP(H52L), CcP(H52Q), and CcP(R48L/W51L/H52L) are -189, -170, -224, and -146mV, respectively. A heme-linked ionization in the reduced enzyme affects the reduction potential for yCcP and all three mutants. Apparent pK(A) values for the heme-linked ionization are 7.5±0.2, 6.5±0.3, 6.4±0.2, and 7.0±0.3 for yCcP and the H52L, H52Q, and R48L/W51L/H52L mutants, respectively. A cooperative, two-proton ionization causing a spectroscopically-detectable transition was observed in the ferrous states of yCcP, CcP(H52L) and CcP(H52Q), with apparent pK(A) values of 7.7±0.2, 7.4±0.1 and 7.8±0.1, respectively. These data indicate that: (1) the distal histidine in CcP is not the site of proton binding upon reduction of the ferric CcP, (2) the distal histidine is not one of the two groups involved in the cooperative, two-proton ionization observed in ferrous CcP, and (3) the proton-binding site is not involved in the cooperative, two-proton ionization observed in the reduced enzyme.

SUBMITTER: DiCarlo CM

PROVIDER: S-EPMC3071893 | biostudies-literature | 2011 Apr

REPOSITORIES: biostudies-literature

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Reduction potential of yeast cytochrome c peroxidase and three distal histidine mutants: dependence on pH.

DiCarlo Cory M CM Vitello Lidia B LB Erman James E JE

Journal of inorganic biochemistry 20110109 4

The pH dependence of the Fe(III) reduction potential, E(0)', for yeast cytochrome c peroxidase (yCcP) and three distal pocket mutants, CcP(H52L), CcP(H52Q), and CcP(R48L/W51L/H52L), has been determined between pH 4 and 8. E(0)' values at pH 7.0 for the yCcP, CcP(H52L), CcP(H52Q), and CcP(R48L/W51L/H52L) are -189, -170, -224, and -146mV, respectively. A heme-linked ionization in the reduced enzyme affects the reduction potential for yCcP and all three mutants. Apparent pK(A) values for the heme-l ...[more]

PMID: 21334283

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Project description:Cytochrome-c-peroxidase (CCP) contains a five-coordinate heme active site. The reduction potential for the ferric to ferrous couple in CCP is anomalously low and pH dependent (Eo = ~-180 mV vs. S.H.E. at pH 7). The contribution of the protein environment to the tuning of the redox potential of this couple is evaluated using site directed mutants of several amino acid residues in the environment of the heme. These include proximal pocket mutation to residues Asp-235, Trp-191, Phe-202 and His-175, distal pocket mutation to residues Trp-51, His-52, and Arg-48; and a heme edge mutation to Ala-147. Where unknown, the structural changes resulting from the amino acid substitution have been studied by X-ray crystallography. In most cases, ostensibly polar or charged residues are replaced by large hydrophobic groups or alternatively by Ala or Gly. These latter have been shown to generate large, solvent filled cavities. Reduction potentials are measured as a function of pH by spectroelectrochemistry. Starting with the X-ray derived structures of CCP and the mutants, or with predicted structures generated by Molecular Dynamics (MD), predictions of redox potential changes are modeled using the Protein Dipoles Langevin Dipoles (PDLD) method. These calculations serve to model an electrostatic assessment of the redox potential change with simplified assumptions about heme iron chemistry, with the balance of the experimentally observed shifts in redox potential being thence attributed to changes in the ligand set and heme coordination chemistry, and/or other changes in the structure not directly evident in the X-ray structures (e.g. ionization states, specific roles played by solvent species, or conformationally flexible portions of the protein). Agreement between theory and experiment is good for all mutant proteins with the exception of the mutation Arg 48 to Ala, and His 52 to Ala. In the former case, the influence of phosphate buffer is adduced to account for the discrepancy, and measurements made in a bis-tris propane/2-(N-morpholino)ethanesulfonic acid buffer system agree well with theory. For the latter case, an unknown structural element relevant to His-52, and/or solvent influence in the mutant akin to anion binding in the distal pocket (though lacking proof that it is) manifests in this mutant. The use of exogenous (sixth) ligands in dissecting the contributions to control of redox potential are also explored as a pathway for model building.

Dataset Information

Reduction potential of yeast cytochrome c peroxidase and three distal histidine mutants: dependence on pH.

Publications

Reduction potential of yeast cytochrome c peroxidase and three distal histidine mutants: dependence on pH.

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