Kinetics of intramolecular electron transfer in cytochrome bo₃ from Escherichia coli

We have examined the temperature dependence of the intramolecular electron transfer (ET) between heme b and heme o₃ in CO-mixed valence cytochrome bo₃ (Cbo) from Escherichia coli. Upon photolysis of CO-mixed valence Cbo rapid ET occurs between heme o₃ and heme b with a rate constant of 2.2 × 10⁵ s^-1 at room temperature. The corresponding rate of CO recombination is found to be 86 s^-1. From Eyring plots the activation energies for these two processes are found to be 3.4 kcal/mol and 6.7 kcal/mol for the ligand binding and ET reactions, respectively. Using variants of the Marcus equation the reorganization energy (λ), electronic coupling factor (H_AB), and the ET distance were found to be 1.4 ± 0.2 eV, (2 ± 1) × 10^-3 eV, and 9 ± 1 Å, respectively. These values are quite distinct from the analogous values previously obtained for bovine heart cytochrome c oxidase (CcO) (0.76 eV, 9.9 × 10^-5 eV, 13.2 Å). The differences in mechanisms/pathways for heme b/heme o₃ and heme a/heme a₃ ET suggested by the Marcus parameters can be attributed to structural changes at the Cu_B site upon change in oxidation state as well as differences in electronic coupling pathways between Heme b and heme o₃.