Intramolecular Electron Transfer in Cytochrome b₅ Labeled with Ruthenium(II) Polypyridine Complexes: Rate Measurements in the Marcus Inverted Region

Three derivatives of recombinant T65C-cytochrome b₅ were produced by labeling the single sulfhydryl group at Cys-65 with (4-(bromomethyl)-4′-methylbipyridine) bis (bipyridine) ruthenium (2+), (4-bromomethyl-4′-methylbipyridine) bis (4,4′-dimethylbipyridine) ruthenium(2+), and (4-(bromomethyl)-4′-methylbipyridine) (bipyrimidine)-(bipyridine) ruthenium(2+). The ruthenium labels are linked to the heme iron through a well-defined 12-covalent-bond path and provide a range of free energies of reaction which bracket the expected reorganizational energy of 0.94 eV. The rate constants for photoinduced electron transfer from Ru(II*) to the ferric heme were 1.4 × 10⁷, 1.7 × 10⁷, and 6 × 10⁵ s⁻¹, respectively, for the three different labeled proteins. The rate constants for the thermal back-reaction from the ferrous heme to Ru (III) were 6.0 × 10⁶, 5.4 × 10⁶, and 2.3 × 10⁶ s⁻¹, respectively. The rate constants show an inverse dependence on driving force, as predicted by Marcus (Marcus, R. A. J. Chem. Phys. 1957, 26, 867–871), with an electronic coupling term consistent with parameters for covalent bond coupling suggested by Beratan et al. (Beratan, D. N.; Onuchic, J. N.; Betts, J. N.; Bowler, B. E.; Gray, H. B. J. Am. Chem. Soc. 1990, 112, 7915–7920). The electronic coupling is also consistent with a simple exponential decay model with β = 1.4 Å and a through space separation of 12 Å. The rate constants were independent of temperature over the range +25 to −90 °C.