Liang Zhao-Xun, Kurnikov Igor V, Nocek Judith M, Mauk A Grant, Beratan David N, Hoffman Brian M
Department of Chemistry, Northwestern University, 2145 Sheridan Road, Evanston, Illinois 60208, USA.
J Am Chem Soc. 2004 Mar 10;126(9):2785-98. doi: 10.1021/ja038163l.
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.
马肌红蛋白（Mb）为探究静电作用对动态[Mb，细胞色素b(5)]电子转移（ET）复合物中结合和反应活性的影响提供了一个便利的“实验平台”。我们结合了诱变和血红素中和技术，制备了一组六个Mb表面电荷变体：[S92D]Mb和[V67R]Mb突变体在“正面”引入了额外电荷，并且将血红素二酯掺入其中每个变体中，中和了血红素丙酸酯上的电荷，这进一步增加了“正面”的正电荷。对于这组突变体，相对于天然Mb，Mb的名义电荷变化了-1至+3个单位。对于该组中的每个成员，我们测量了光引发的(3)ZnDMb→Fe(3+)b(5)ET反应的双分子猝灭速率常数（k(2)）作为离子强度的函数。我们发现：（i）结合与反应活性之间存在显著解耦，其中在Mb组内k(2)变化约10(3)倍，而结合亲和力没有显著变化；（ii）ET反应发生在“动态对接”模型的“热力学”或“快速交换”极限内，其中大量弱结合的蛋白质-蛋白质构型对结合有贡献，但只有少数是有反应活性的，这一事实表明零离子强度下的双分子速率常数随Mb上的净电荷呈指数变化；（iii）布朗动力学对接图谱使我们能够可视化动态对接的微观基础。为了描述这些结果，我们提出了一种新的理论方法，该方法在蒙特卡罗（MC）采样框架中，将途径供体/受体耦合计算与基于泊松-玻尔兹曼的对接能量静电估计进行数学结合，因此特别适用于分子间ET问题。该程序极其高效，因为它通过在计算本身中引入“反应活性过滤器”，而不是作为后续步骤，仅针对功能活性复合物几何结构。这种效率使我们能够采用计算成本更高且更准确的方法来描述相关的分子间相互作用能和蛋白质介导的供体/受体耦合相互作用。在此用于计算Mb和cyt b(5)之间ET的双分子速率常数在肌红蛋白表面电荷、pH值和离子强度变化时的变化。
