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中心地位。然而，电子/空穴在蛋白质-蛋白质界面上的转移几乎总是不受ET过程本身的控制，而是由界面结构集合之间和内部的构象转换动力学控制。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。在目前的研究中，我们认识到锌取代的血红蛋白及其氧化还原伙伴之间的闪光诱导的蛋白质-蛋白质光循环的“前进”和“后退”步骤涉及不同的初始构型集合，因此应该对构型相互转换的调制做出不同的反应。这些“差分动力学”的测量将在我们的研究中发挥重要作用。他们将用动力学-动力学（KD）模型进行分析，该模型首次结合了光周期内ET与构型动力学的耦合（第D1节）。我们通过研究三个表现出互补动态特征的系统来实现我们的目标。KD模型的实施已经开始测量结构定义的混合金属[Zn；血红蛋白（Hb）杂交体。我们的假设是，表面上刚性的[Zn，Fe]杂化体监测到的ET光循环与[?1(Zn);？2(Fe)] ET伙伴（第D2节）。

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.