Electrostatic control of protein docking and electron transfer between myoglobin and cytochrome b(5)

The interaction of trypsin-digested bovine cytochrome b ₅ (cyt b₅) with horse heart myoglobin (Mb) and the inter protein electron transfer (ET) between these redox partners have been studied to gain better understanding of ET processes between weakly bound protein partners. The formation of a protein-protein complex has been confirmed and the binding affinities of metMb and zinc-porphyrin subsititued Mb (ZnMb) for cyt b5 have been measured independently by NMR and isothermal calorimetry methods. While ET pathway calculations suggest that only a small, narrow region of the Mb surface is ET reactive, Brownian dynamic (BD) simulations indicate that cyt b₅ binds over a broad surface of Mb that includes its heme edge, and that the overall ET rate is determined by a small set of energetically unfavorable complex conformations. These results are interpreted with a dynamic docking model which proposes that Mb binds cyt b₅ in a large ensemble of protein binding conformations, but that only a small subset is ET reactive.; We have found that surface charge modification by site-directed mutagenesis and heme propionate modification can greatly change the overall second-order rate of the photoinduced ET, (3ZnP)Mb + Fe3+cyt b₅ → (ZnP)+Mb + Fe2+cyt b₅, without significantly perturbing Mb's affinity for cyt b₅. To rationalize these results, we have proposed that ET occurs only (primarily) when cyt b₅ binds within a small area of the surface near the Mb heme edge and involves only a small subset of highly ET reactive conformations. The probability of these conformations is enhanced by the optimization of complementary electrostatic interactions without substantial influence on the binding affinity. These results support the idea that in this, and possibly other, electrostatic interactions play a more important role in directing ‘productive’ protein docking than in contributing to the overall binding energy. Therefore, by optimization of electrostatic interaction between interacting proteins, one can decouple the issue of rapid reaction, in this case fast interprotein ET, from that of binding affinity. This issue is important because high binding affinities are undesirable for fast interprotein ET or other protein interactions where high turn-over is required.