Dynamic docking and electron transfer between myoglobin and cytochrome b5

The interaction of trypsin-digested bovine cytochrome b(5) (cyt b(5)) 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 (k(2)) for photoinduced ET between zinc-substituted Mb (ZnMb) and cyt b(5) 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 b(5) have been measured by two techniques: H-1 NMR titrations at pH 6.0 give binding constants of K(a)approximate to(1.0+/-0)x10(3) M-1 for netMb and K(a)approximate to(0.75+/-0.1)x10(3) M-1 for ZnMb; isothermal calorimetry gives K(a)approximate to(0.35+/-0.1)x10(3) M-1 for ZnMb. Brownian dynamic (BD) simulations show that cyt b(5) binds over a broad surface of Mb that includes its heme edge. The experimental results arc described in terms of a dynamic docking model which proposes that Mb binds cyt b(5) 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 k(2) decreases with increasing pH: increasing pH not only weakens the binding affinity but also reduces the number of binding conformations with high ET reactivity.