No abstract

Horse myoglobin (Mb) provides a convenient "workbench" for probing the effects of electrostatics on binding and reactivity in the dynamic [Mb, cytochrome b(5)] electron-transfer (ET) complex. We have combined mutagenesis and heme neutralization to prepare a suite of six Mb surface-charge variants: the [S92D]Mb and [V67R]Mb mutants introduce additional charges on the "front" face, and incorporation of the heme di-ester into each of these neutralizes the charge on the heme propionates which further increases the positive charge on the "front" face. For this set of mutants, the nominal charge of Mb changes by -1 to +3 units relative to that for native Mb. For each member of this set, we have measured the bimolecular quenching rate constant (k(2)) for the photoinitiated (3)ZnDMb --> Fe(3+)b(5) ET reaction as a function of ionic strength. We find: (i) a dramatic decoupling of binding and reactivity, in which k(2) varies approximately 10(3)-fold within the suite of Mbs without a significant change in binding affinity; (ii) the ET reaction occurs within the "thermodynamic" or "rapid exchange" limit of the "Dynamic Docking" model, in which a large ensemble of weakly bound protein-protein configurations contribute to binding, but only a few are reactive, as shown by the fact that the zero-ionic-strength bimolecular rate constant varies exponentially with the net charge on Mb; (iii) Brownian dynamic docking profiles allow us to visualize the microscopic basis of dynamic docking. To describe these results we present a new theoretical approach which mathematically combines PATHWAY donor/acceptor coupling calculations with Poisson-Boltzmann-based electrostatics estimates of the docking energetics in a Monte Carlo (MC) sampling framework that is thus specially tailored to the intermolecular ET problem. This procedure is extremely efficient because it targets only the functionally active complex geometries by introducing a "reactivity filter" into the computations themselves, rather than as a subsequent step. This efficiency allows us to employ more computationally expensive and accurate methods to describe the relevant intermolecular interaction energies and the protein-mediated donor/acceptor coupling interactions. It is employed here to compute the changes in the bimolecular rate constant for ET between Mb and cyt b(5) upon variations in the myoglobin surface charge, pH, and ionic strength.

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.

The transient complex of bovine myoglobin and cytochrome b(5) has been investigated using a combination of NMR chemical shift mapping, (15)N relaxation data, and protein docking simulations. Chemical shift perturbations observed for cytochrome b(5) amide resonances upon complex formation with either metmyoglobin (Fe(III)) or carbon monoxide-bound myoglobin (Fe(II)) are more than 10-fold smaller than in other transient redox protein complexes. From (15)N relaxation experiments, an increase in the overall correlation time of cytochrome b(5) in the presence of myoglobin is observed, confirming that complex formation is occurring. The chemical shift perturbations of proton and nitrogen amide nuclei as well as heme protons of cytochrome b(5) titrate with increasing myoglobin concentrations, also demonstrating the formation of a weak complex with a K(a) in the inverse millimolar range. The perturbed residues map over a wide surface area of cytochrome b(5), with patches of residues located around the exposed heme 6-propionate as well as at the back of the protein. The nature of the affected residues is mostly negatively charged contrary to perturbed residues in other transient complexes, which are mainly hydrophobic or polar. Protein docking simulations using the NMR data as constraints show several docking geometries both close to and far away from the exposed heme propionates of myoglobin. Overall, the data support the emerging view that this complex consists of a dynamic ensemble of orientations in which each protein constantly diffuses over the surface of the other. The characteristic NMR features may serve as a structural tool for the identification of such dynamic complexes.

To characterize the electrostatic complex formed between myoglobin (Mb) and cytochrome b 5 (Feb 5), we have performed flash photolysis triplet-quenching and electron-transfer (ET) measurements of the interaction between Zn deuteroporphyrin (ZnD)-substituted Mb (sperm whale) (ZnDMb) and Feb 5(trypsin-solubilized, bovine) at pH values between 6 and 7.5. For pH values between pH 6 and pH 7.5, the quenching rate constant (Δk) varies linearly with [Fe3+ b 5]. The slope (M) obtained from plots of Δk versus [Fe3+ b 5] is strongly dependent on pH (M = 140 × 106 M-1 s-1 at pH 6 and M = 2.4 × 106 M-1 s-1 at pH 7.5). The triplet decay profiles remain exponential throughout these titrations. Together, these results indicate that the association constant obeys the inequality, Ka ≤ 3000 M-1 and that the lower limit for the rate constant for dissociation of the 3 DA complex of (k off)min = 106 s-1 at pH 6 and (k off)min = 104 s-1 at pH 7.5. Transient absorption measurements have shown that this quenching of 3ZnDMb by Fe3+ b 5 can be attributed to intracomplex 3ZnD → Fe3+P ET and that the transient absorbance changes observed at the 3 D/D isosbestic points represent the time evolution of the ( D + A - ), [ZnD+Mb, Fe2+ b 5] intermediate, I. The long-time behavior of the progress curves (t ≥ 20 ms) collected during a titration of Fe3+ b 5 by ZnDMb (reverse titration protocol) is neither purely second-order nor purely first-order but rather resembles a mixed-order process involving both the ( D + A - ) complex and its dissociated components. Modeling this data indicates that the D + A - complex product must dissociate with a rate constant slower than that of the precursor, DA, complex. Theoretical studies of the protein pair by Brownian dynamics simulations show that Mb has a broad reactive surface which encompasses the “hemisphere” that includes the exposed heme edge. The most stable complexes occur when b 5 is bound at one of two subdomains within this hemisphere. The kinetics measurements and calculations taken together allow us to discuss the relative importance of global and local electrostatics in regulating protein−protein recognition and reactivity.

Direct measurements of electron transfer (ET) within a protein-protein complex with a redesigned interface formed by physiological partner proteins myoglobin (Mb) and cytochrome b5 (b5) reveal interprotein ET rates comparable to those observed within the photosynthetic reaction center. Brownian dynamics simulations show that Mb in which three surface acid residues are mutated to lysine binds b5 in an ensemble of configurations distributed around a reactive most-probable structure. Correspondingly, charge-separation ET from a photoexcited singlet zinc porphyrin incorporated within Mb to the heme of b5 and the follow-up charge-recombination exhibit distributed kinetics, with median rate constants, kfs=2.1×109second−1 and kbs=4.3×1010second−1, respectively. The latter approaches that for the initial step in photosynthetic charge separation, k = 3.3 × 1011 second−1.

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...

The physiological electron-transfer (ET) partners, cytochrome c peroxidase (CcP) and cytochrome c (Cc)1, can be modified to exhibit photoinitiated ET through substitution of Zn (or Mg) for Fe in either partner. Laser excitation of the Zn-porphyrin (ZnP) to its triplet excited state (3ZnP) initiates direct heme-heme ET to the ferriheme center of its partner across the protein-protein interface. This photoinitiated ET produces the charge-separated intermediate, I = [ZnP+CcP, Fe2+Cc], with a metalloporphyrin pi-cation radical (ZnP+) in the donor protein and a ferroheme acceptor protein. I, in general, is thought to return to the ground state by a thermal ET process that involves direct heme-heme back-ET to complete a simple photocycle. We here contrast intracomplex ET between yeast iso-1 Cc and ZnCcP(WT) (wild-type) with that for two ZnCcP(X) variants: X = W191F, with redox-active W191 replaced by Phe; WYM4, a W191F mutant with further replacement of four other potentially redox-active sites (W51F, Y187F, Y229F, and Y236F). The results show that W191 acts as an ET mediator, which "short-circuits" the direct heme-heme back-ET through a two-step, hopping process in which the ZnP+ cation radical formed by photoinitiated ET rapidly oxidizes W191, and the resultant W191+, in turn, rapidly oxidizes Fe2+Cc.