Electron Transfer within Protein Complexes

描述（申请人提供）：远程电子转移（ET）的研究在现代生物学和化学中占有核心地位。然而，跨蛋白质-蛋白质界面的电子/空穴的转移几乎总是不受ET过程本身的控制，而是受界面构型之间和内部集合的构象转换动力学的控制。我们的总体目标是了解蛋白质间ET如何与这些运动耦合并受其控制。在本期间，我们认识到，闪光诱导的蛋白质-蛋白质光循环的ET之间的锌取代血红素蛋白和它的氧化还原伙伴的“向前”和“向后”的步骤涉及不同的初始构型系综，因此应该不同地响应配置相互转换的调制。这些“微分动力学”的测量将在我们的研究中发挥重要作用。他们将分析与动力学动力学（KD）模型，其中纳入了第一次耦合的ET内的光循环（第D1节）的构型动力学。我们追求我们的目标，通过研究三个系统，表现出互补的动态特性。KD模型的实施已经开始与结构上定义的，混合金属，[Zn; Fe]血红蛋白（Hb）的杂合体内的亚基间ET的测量。这是我们的假设，ET光循环监测显然是刚性的[锌，铁]杂化物耦合到动态之间的接口[？1（Zn）;？2（Fe）] ET伴侣（第D2节）。 锌-细胞色素c过氧化物酶（CcP）和细胞色素c（Cc）之间紧密结合的1：1复合物内的ET光循环探测了ET与能量和时间尺度层次上的动力学的耦合（第C1，2节; D3）。我们假设，ET附近的环境是由几个结构之间的转换占主导地位，但这'冻结'后冷却到T ~ 0 C和/或添加小溶质;过渡内的子状态，包括低温结构的合奏，然后冻结在一个合作的构象转变的范围内，220- 250 K。 肌红蛋白（Mb）和细胞色素b 5（cytb 5）的复合物（第C1、3节;第D4节）是对其他两个系统的补充，因为它们表现出一系列结构，其中只有少数是反应性的（“动态对接; DD）。这导致了意想不到的ET行为，并为我们的目标提供了一个明显不同的演员。Mb电荷交换突变加强结合，足以将复合物移出DD方案并进入具有强1：1结合的方案，并且这导致3 ZnMb的显著快速的复合物内猝灭。我们将（i）研究这个过程的物理学和动力学;（ii）探索ET/动力学耦合如何随着具有多个结合结构的DD复合物通过离子强度变化和/或突变调节亲和力转换为1：1结合而变化;以及（iii）利用ET活性复合物的结合自由能的线性自由能分解，同时实验测试其基本基础和解释。

DESCRIPTION (provided by applicant): The study of long-range electron transfer (ET) occupies a central place in modern biology and chemistry. However, the transfer of an electron/hole across a protein-protein interface almost invariably is controlled not by the ET process itself, but by the dynamics of conformational conversion among and within ensembles of interface configurations. Our overall aim is to understand how inter-protein ET is coupled to and controlled by these motions. During the current period we recognized that the 'forward' and 'back' steps of a flash- induced protein-protein photocycle of ET between a zinc-substituted hemoprotein and its redox partner involve different initial configurational ensembles, and hence should respond differently to the modulation of configuration interconversion. Measurements of these 'differential dynamics' will play an important role in our studies. They will be analyzed with a kinetic-dynamic (KD) model which incorporates for the first time the coupling of ET to configurational dynamics within a photocycle (Section D1). We pursue our aims through studies of three systems which exhibit complementary dynamic characteristics. Implementation of the KD model has begun with the measurements of inter-subunit ET within the structurally defined, mixed-metal, [Zn; Fe] hemoglobin (Hb) hybrids. It is our hypothesis that the ET photocycle monitored for the apparently rigid [Zn,Fe] hybrids is coupled to dynamics at the interface between [?1(Zn);?2(Fe)] ET partners (Section D2). The ET photocycle within the tightly-bound 1:1 complex between Zn-cytochrome c peroxidase (CcP) and cytochrome c (Cc) probes the coupling of ET to dynamics on a hierarchy of energy and time scales (Sections C1,2; D3). We hypothesize that ET near ambient is dominated by conversion between a few structures, but this 'freezes out' upon cooling to T ~0C and/or the addition of small solutes; transitions within the ensemble of substates that comprise the low-temperature structure then freeze out during a cooperative conformational transition in the range, 220-250K. Complexes of myoglobin(Mb) and cytochromeb5 (cytb5)(SectionsC1,3;D4)complement the other two systems in that they exhibit an ensemble of structures, only a few of which are reactive ('Dynamic Docking; DD). This leads to unexpected ET behaviors and gives a distinctly different cast to our aims. Mb charge- exchange mutations strengthen binding enough to shift the complex out of the DD regime and into a regime with strong 1:1 binding, and this results in remarkably fast intra-complex quenching of 3ZnMb. We shall (i) study the photophysics and dynamics of this process; (ii) explore how the ET/dynamics coupling changes as a DD complex with multiple bound structures is converted to 1:1 binding by tuning affinities through ionic strength variation and/or mutations; and (iii) exploit a linear free energy decomposition of the binding free energy of ET-active complexes, while experimentally testing its fundamental basis and interpretation.