Dynamic docking and electron transfer between myoglobin and cytochrome b₅

The interaction of trypsin-digested bovine cytochrome b₅ (cyt b₅) with horse heart myoglobin (Mb) and the interprotein electron transfer (ET) between these redox partners have been studied to gain better understanding of ET processes between weakly bound protein partners. The bimolecular rate constant (k₂) for photo-induced ET between zinc-substituted Mb (ZnMb) and cyt b₅ decreases with increasing ionic strength, consistent with the predominantly electrostatic character of this complex. The formation of a protein-protein complex has been confirmed and the binding affinities of metMb and ZnMb for cyt b₅ have been measured by two techniques: ¹H NMR titrations at pH 6.0 give binding constants of K_a≈(1.0±0.1)×10³ M^-1 for metMb and K_a≈(0.75±0.1)×10³ M^-1 for ZnMb; isothermal calorimetry gives K_a≈(0.35±0.1)×10³ M^-1 for ZnMb. Brownian dynamic (BD) simulations show that cyt b₅ binds over a broad surface of Mb that includes its heme edge. The experimental results are described in terms of a dynamic docking model which proposes that Mb binds cyt b₅ in a large ensemble of protein binding conformations, not one or a few dominant ones, but that only a small subset are ET reactive. Aided by the BD simulations, this model explains why k₂ decreases with increasing pH: increasing pH not only weakens the binding affinity but also reduces the number of binding conformations with high ET reactivity.