Impact of proximal and distal pocket site-directed mutations on the ferric/ferrous heme redox potential of yeast cytochrome c peroxidase

Autor: G. M. Jensen, D. B. Goodin
Rok vydání: 2011
Předmět:
Zdroj: Theoretical Chemistry Accounts. 130:1185-1196
ISSN: 1432-2234
1432-881X
Popis: 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 of residues Asp-235, Trp-191, Phe-202, and His-175, distal pocket mutation of residues Trp-51, His-52, and Arg-48; and a heme edge mutation of 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, with evidence for phosphate binding in the distal pocket, 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, and in this case lacking a phosphate effect) manifests in this mutant. The use of exogenous (sixth) ligands in dissecting the contributions to control of redox potential is also explored as a pathway for model building.
Databáze: OpenAIRE
Externí odkaz: