Symmetrized photoinitiated electron flow within the [Myoglobin:Cytochrome b ₅] complex on singlet and triplet time scales: Energetics vs dynamics

We report here that photoinitiated electron flow involving a metal-substituted (M = Mg, Zn) myoglobin (Mb) and its physiological partner protein, cytochrome b₅ (cyt b₅) can be "symmetrized": the [Mb:cyt b₅] complex stabilized by three D/E → K mutations on Mb (D44K/D60K/E85K, denoted MMb) exhibits both oxidative and reductive ET quenching of both the singlet and triplet photoexcited MMb states, the direction of flow being determined by the oxidation state of the cyt b₅ partner. The first-excited singlet state of MMb (¹MMb) undergoes ns-time scale reductive ET quenching by Fe²⁺cyt b₅ as well as ns-time scale oxidative ET quenching by Fe³⁺cyt b₅, both processes involving an ensemble of structures that do not interconvert on this time scale. Despite a large disparity in driving force favoring photooxidation of ¹MMb relative to photoreduction (δ(-ΔG⁰) ≈ 0.4 eV, M = Mg; ≈ 0.2 eV, M = Zn), for each M the average rate constants for the two reactions are the same within error, ¹k_f > 10⁸ s^-1. This surprising observation is explained by considering the driving-force dependence of the Franck-Condon factor in the Marcus equation. The triplet state of the myoglobin (³MMb) created by intersystem crossing from ¹MMb likewise undergoes reductive ET quenching by Fe²⁺cyt b₅ as well as oxidative ET quenching by Fe³⁺cyt b₅. As with singlet ET, the rate constants for oxidative ET quenching and reductive ET quenching on the triplet time scale are the same within error, ³k_f ≈ 10⁵ s^-1, but here the equivalence is attributable to gating by intracomplex conversion among a conformational ensemble.