EFFECTS OF MUTATIONS AND COMPLEX-FORMATION ON THE REDUCTION POTENTIALS OF CYTOCHROME-C AND CYTOCHROME-C PEROXIDASE

A continuum model is used to calculate the effects of point mutations and complex formation on the reduction potentials of yeast iso-1-cytochrome c (cc) and yeast cytochrome c peroxidase (CCP). In this model a protein is represented by a low dielectric region embedded in the high dielectric solvent. Qualitative analysis shows that the model can account for a wide range of factors that determine the reduction potential. These include the charge and polarity of a surface residue, the polarity of an interior residue, and the size of a residue which controls the exposure of the heme to the solvent. The continuum model allows for a reasonably good reproduction of a demanding set of data on cc, consisting of the measured differences in reduction potential between the wild type and seven mutants (Arg38 --> Lys, His, Asn, and Ala, Tyr48 --> Phe, Asn52 --> Ile, and Phe82 --> Ser). In the case of CCP, continuum-model calculations on the effects of mutating Asp235 lead to the following conclusions: (1) The imidazolate character of wild-type His175, shown by resonance Raman spectroscopy and NMR, is critical in lowering the reduction potential of the wild type 70 mV from that of the Glu235 mutant, which has the same charge as the wild type. (2) A sixth ligand, such as a water molecule, is necessary for maintaining the reduction potential of the Ala235 mutant at a level that is only 35 mV above the reduction potential of the Glu235 mutant, which has an extra buried carboxylate. (3) That the Asn235 and Ala235 mutants have almost equal reduction potentials is due to the fact that the amide dipole of the Asn235 residue is oriented such that it does not stabilize or destabilize the charge on the heme. In contrast to previous expectations, complex formation is found to have only a small effect on the reduction potential of cc and no effect at all on that of CCP. The protein matrices are found to play an important role of reducing the outer reorganization energy from what would have been if the redox centers were embedded directly in the solvent and thus speeding up the electron transfer. By relating electron transfer to redox reactions, a method for obtaining the inner reorganization energy is proposed.