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-cytoc hrome 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 acc ount for a wide range of factors that determine the reduction potentia l. These include the charge and polarity of a surface residue, the pol arity of an interior residue, and the size of a residue which controls the exposure of the heme to the solvent. The continuum model allows f or a reasonably good reproduction of a demanding set of data on cc, co nsisting of the measured differences in reduction potential between th e wild type and seven mutants (Arg38 --> Lys, His, Asn, and Ala, Tyr48 --> Phe, Asn52 --> Ile, and Phe82 --> Ser). In the case of CCP, conti nuum-model calculations on the effects of mutating Asp235 lead to the following conclusions: (1) The imidazolate character of wild-type His1 75, shown by resonance Raman spectroscopy and NMR, is critical in lowe ring the reduction potential of the wild type 70 mV from that of the G lu235 mutant, which has the same charge as the wild type. (2) A sixth ligand, such as a water molecule, is necessary for maintaining the red uction potential of the Ala235 mutant at a level that is only 35 mV ab ove the reduction potential of the Glu235 mutant, which has an extra b uried 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 des tabilize the charge on the heme. In contrast to previous expectations, complex formation is found to have only a small effect on the reducti on potential of cc and no effect at all on that of CCP. The protein ma trices are found to play an important role of reducing the outer reorg anization energy from what would have been if the redox centers were e mbedded directly in the solvent and thus speeding up the electron tran sfer. By relating electron transfer to redox reactions, a method for o btaining the inner reorganization energy is proposed.