Effect of Binding Cytochrome c and Ionic Strength on the Reorganizational Energy and Intramolecular Electron Transfer in Cytochrome b₅ Labeled with Ruthenium(II) Polypyridine Complexes

The rates of intramolecular electron transfer in T6SC cytochrome b₅ labeled with three different ruthenium polypyridine complexes at Cys-65 have been measured over a range of ionic strengths in the presence and absence of cytochrome c. The redox potentials of the ruthenium complexes provide for a range of free energies of reaction which span the reorganizational energy of the system. The system obeys the familiar dependence of In k_obs on free energy of reaction predicted by Marcus (Marcus, R. A. J. Chem. Phys. 1956,24,966). The reorganization energies for these reactions obtained at 0 and 300 mM NaCl in the absence of cytochrome c are 1.02 and 0.93 eV, respectively. The corresponding electronic coupling elements calculated as H_ab from k_max = 2.0 × 10⁷ and 1.4 × 10⁷ s^-1are 0.29 and 0.21 cm^-1, respectively. Surprisingly, the rate constants obtained with cytochrome c bound to cytochrome b₅ ([NaCl] = 0) are the same as those obtained at [NaCl] > 150 mM in the absence of cytochrome c. These rate constants are, however, very different from those obtained at low [NaCl] in the absence of cytochrome c. We suggest that these results can be explained in terms of an alteration of the solvent reorganizational barrier either by binding of cytochrome c and exclusion of water from the binding domain or by the influence of sodium and chloride ions on the solvent in close proximity to the redox centers.