Dynamic Docking and Electron Transfer between Zn-myoglobin and Cytochrome b₅

Abstract

We present a broad study of the effect of neutralizing the two negative charges of the Mb propionates on the interaction and electron transfer (ET) between horse Mb and bovine cyt b(5), through use of Zn-substituted Mb (ZnMb, 1) to study the photoinitiated reaction, ((3)ZnP)Mb + Fe(3+)cyt b(5) --> (ZnP)(+)Mb + Fe(2+)cyt b(5). The charge neutralization has been carried out both by replacing the Mb heme with zinc-deuteroporphyrin dimethylester (ZnMb(dme), 2), which replaces the charges by small neutral hydrophobic patches, and also by replacement with the newly prepared zinc-deuteroporphyrin diamide (ZnMb(diamide), 3), which converts the charged groups to neutral, hydrophilic ones. The effect of propionate neutralization on the conformation of the zinc-porphyrin in the Mb heme pocket has been studied by multinuclear NMR with an (15)N labeled zinc porphyrin derivative (ZnMb((15)N-diamide), 4). The rates of photoinitiated ET between the Mb's (1-3) and cyt b(5) have been measured over a range of pH values and ionic strengths. Isothermal titration calorimetry (ITC) and NMR methods have been used to independently investigate the effect of charge neutralization on Mb/b(5) binding. The neutralization of the two heme propionates of ZnMb by formation of the heme diester or, for the first time, the diamide increases the second-order rate constant of the ET reaction between ZnMb and cyt b(5) by as much as several 100-fold, depending on pH and ionic strength, while causing negligible changes in binding affinity. Brownian dynamic (BD) simulations and ET pathway calculations provide insight into the protein docking and ET process. The results support a new "dynamic docking" paradigm for protein-protein reactions in which numerous weakly bound conformations of the docked complex contribute to the binding of cyt b(5) to Mb and Hb, but only a very small subset of these are ET active, and this subset does not include the conformations most favorable for binding; the Mb surface is a large "target" with a small "bullseye" for the cyt b(5) "arrow". This paradigm differs sharply from the more familiar, "simple" docking within a single, or narrow range of conformations, where binding strength and ET reactivity increase in parallel. Likewise, it is distinct from, although complementary to, the well-known picture of conformational control of ET through "gating", or a related picture of "conformational coupling". The new model describes situations in which tight binding does not correlate with efficient ET reactivity, and explains how it is possible to modulate reactivity without changing affinity. Such "decoupling" of reactivity from binding clearly is of physiological relevance for the reduction of met-Mb in muscle and of met-Hb in a red cell, where tight binding of cyt b(5) to the high concentration of ferrous-Mb/Hb would prevent the cytochrome from finding and reducing the oxidized proteins; it likely is of physiological relevance in other situations, as well.

Differential influence of dynamic processes on forward and reverse electron transfer across a protein-protein interface

“…Unlike the hybrids, results to date suggest that intracomplex ET may not persist as the viscosity increases without bound (38). (4,14). These studies showed that the bimolecular, 3 ZnDMb 3 Fe 3ϩ b 5 forward ET rate constant, k f2 , increases dramatically as the positive charge on Mb is changed by the nominal amounts, Ϫ1 Յ ⌬q nom Յ ϩ3, but without a substantial increase in the binding affinity (4).…”

“…Samples for ET studies were prepared in the dark under a N 2 atmosphere as described (4,14). Briefly, protein stock solutions for flash photolysis were exchanged into working buffers: 10 For the experiments with the Hb hybrid and the [ZnCcP(W191F),Fe 3ϩ Cc] complex, cryosolvents of varying viscosity were made at a selected (vol͞vol) composition of glycerol and deionized water.…”

“…1 A Right (4,14). This paradigm in protein-protein ET was revealed through the decoupling of binding and reactivity: increasing the surface charge on Mb caused the second-order rate for ET from 3 ZnMb to Fe 3ϩ b 5 to increase by nearly three orders of magnitude without significantly varying Mb's binding affinity for Fe 3ϩ b 5 .…”

“…Recent studies of ET between myoglobin (Mb) and cytochrome b 5 (Fe 3ϩ b 5 ), which bind to each other by weak electrostatic interactions, disclosed a new dynamic docking paradigm in protein-protein reaction dynamics: the landscape involves numerous configurations of similar affinity, only a subset of which is active in ET ( Fig. 1 A Right) (4,14).…”

Interprotein electron transfer from cytochromec2to photosynthetic reaction center: Tunneling across an aqueous interface

“…This prediction is confirmed by the finding (17) that k 2 is independent of the unimolecular ET rate in the bound state, k e . Note that the opposite fast exchange reaction regime, k off Ͼ Ͼ k e , in which the second-order rate constant is given by k 2 Ϸ k e ͞K D , has been observed in other cases (13,18). More recently, structural information from the x-ray crystal structure of the cyt:RC complex (14), experiments on mutated RCs (12,19), and electrostatic calculations (20,21) have provided the basis for a more detailed reaction scheme that includes an electrostatically stabilized encounter complex (EC) that is formed before reaction to form the bound state.…”