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

Abstract

We propose that the forward and reverse halves of a flash-induced protein-protein electron transfer (ET) photocycle should exhibit differential responses to dynamic interconversion of configurations when the most stable configuration is not the most reactive, because the reactants exist in different initial configurations: the flash-photoinitiated forward ET process begins with the protein partners in an equilibrium ensemble of configurations, many of which have little or no reactivity, whereas the reactant of the thermal back ET (the charge-separated intermediate) is formed in a nonequilibrium, ''activated'' protein configuration. We report evidence for this proposal in measurements on (i) mixed-metal hemoglobin hybrids, (ii) the complex between cytochrome c peroxidase and cytochrome c, and (iii and iv) the complexes of myoglobin and isolated hemoglobin ␣-chains with cytochrome b 5. For all three systems, forward and reverse ET does respond differently to modulation of dynamic processes; further, the response to changes in viscosity is different for each system. cytochrome c ͉ dynamics ͉ hemoglobin ͉ myoglobin ͉ cytochrome c peroxidase T he long-range transfer of a single electron from donor to acceptor in a condensed phase is a fascinating and widely studied process (1, 2). Much of this work seeks to understand the electron transfer (ET) process itself. However, when the ET event involves a dynamic protein-protein interface, the observed kinetics frequently are controlled not by the ET process itself, but by the dynamics of recognition and binding and͞or conversion within an ensemble of bound configurations (3, 4).Studies of interprotein ET (3, 5-10) began with the implicit assumption of a protein-protein binding-energy landscape with a single reactive complex (Fig. 1A Left), implying a direct correlation between binding and reactivity. When the landscape for complex formation has several discrete minima ( Fig. 1 A Center) the reactive conformation may differ from the most stable one, in which case the observed ET kinetics are controlled by the rates and͞or energetics of conformational conversion within a complex (11-13). 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).A majority of these studies have used a photocycle in which laser-flash excitation of the metallo-porphyrin in a metalsubstituted (M ϭ Zn or Mg) hemoprotein to its triplet excited state ( 3 D) triggers ET from the triplet to the metal center of an acceptor protein (A) across a protein-protein interface, with rate constant, k f , Eq. 1:[1]The acceptor metal center of A typically is a ferri-heme center, Given the exponentially steep fall-off in the matrix element for ET between the two redox centers (19,20), there will be only a subset of conformation...

Long-range electron transfer

“…A critical issue here is understanding bridge energy effects on charge transport through molecular materials, as discussed in this issue of PNAS by Ratner, Wasielewski, and coworkers (100). The role of dynamics in protein ET is another hot topic these days; and two articles in this issue of PNAS report exciting results in this area (47,79). Distant (Ͼ20 Å) charge transport in DNA is still another area of great interest.…”

“…The higher-affinity binding site is the locus for Trp-191 radical reduction by cyt c. Heme (ccp) reduction by cyt c can occur from either the high-or lowaffinity binding site but, when exchange between the two is rapid, reduction from the low-affinity site dominates (77). These studies of ccp͞cyt c and cyt b 5 ͞c ET, including an important contribution by Hoffman and coworkers (79) in this issue of PNAS, have shed much light on the mechanisms of protein-protein ET processes.…”

Distant charge transport

“…As described by Beratan and coworkers (11), these modified proteins now serve as models for studies of the role of polypeptide dynamics on electron tunneling reactions. Electron transfers across protein-protein interfaces have been very actively investigated; articles in this issue by Hoffman and colleagues (12) and by Onuchic and colleagues (13) deal with this subject. Distant redox chemistry also is critically important in DNA, as discussed in the paper by Barton, David, and coworkers (14).…”

Long-range protein electron transfer observed at the single-molecule level: In situ mapping of redox-gated tunneling resonance

“…For example, long-range protein electron transfer (ET) is central in aerobic respiration and photosynthesis. The importance of longrange ET is illustrated by a recent special issue of PNAS on this topic (6)(7)(8)(9)(10)(11)(12). Several articles reflect broadly the current status of this subject.…”