Intramolecular electron-transfer reactions of cytochrome b₅ covalently bonded to ruthenium(II) polypyridine complexes: Reorganizational energy and pressure effects

A series of ruthenium polypyridine complexes have been covalently attached to Cys73 of the T73C variant of synthetic rat liver cytochrome b₅. Photoexcitation of the ruthenium complex results in transient electron transfer from the excited ruthenium complex, Ru(II)*, to the Fe(III) center of the protein followed by a rapid electron-transfer reaction from Fe(II) to Ru(III). The observed rate constants for the excited-state reaction ranged from 3.4 × 10⁷ to 5 × 10⁴ s^-1 and from 4.3 × 10⁷ to 4.5 × 10⁵ s^-1 for the back reaction. The free energy dependence of the rate constants is best described by a reorganizational energy = 1 eV and an electron tunneling matrix element H_AB = 0.35 cm^-1 for electron transfer between the heme iron and the ruthenium complex. The reorganizational energy is the same as that obtained with the T65C variant labeled at Cys65 with the same series of ruthenium complexes. H_AB, however, is 30% larger in the T73C derivatives. The pressure dependence of the rate constants of two derivatives of cytochrome b₅ have been determined in order to investigate the sensitivity of the electronic coupling to pressure dependent van der Waals contacts between the redox centers. The first derivative was made by labeling the T65C variant with Ru((CH₃)₂bpy)₂(CH₃bpyCH ₂-)²⁺ and contains a well defined 12 bond covalent link between the redox centers. The T65C derivative shows no pressure dependence over the range of 0.1-200 MPa. The second derivative was made by labeling the T73C variant with the same complex. In this case, the electronic coupling between the redox centers appears to involve a van der Waals contact between the hydrogens on the γ-carbon of Ser71 and a porphyrin methyl group. This derivative shows a very small negative volume of activation.