The kinetics of flash-induced H+ ion binding by isolated reaction centers (RCs) of Rhodobacter sphaeroides, strain R-26, were measured, using pH indicators and conductimetry, in the presence of terbutryn to block electron transfer between the primary and secondary quinones (QA and QB), and in the absence of exogenous electron donors to the oxidized primary donor, P+, i.e., the P+QA-state. Under these conditions, proton binding by RCs is to the protein rather than to any of the cofactors. After light activation to form P+QA-, the kinetics of proton binding were monoexponential at all pH values studied. At neutral pH, the apparent bimolecular rate constant was close to the diffusional limit for proton transfer in aqueous solution (approximately 10(11) M-1 s-1), but increased significantly in the alkaline pH range (e.g., 2 x 10(13) M-1 s-1 at pH 10). The average slope of the pH dependence was -0.4 instead of -1.0, as might be expected for a H+ diffusion-controlled process. High activation energy (0.54 eV at pH 8.0) and weak viscosity dependence showed that H+ ion uptake by RCs is not limited by diffusion. The salt dependence of the H+ ion binding rate and the pK values of the protonatable amino acid residues of the reaction center implicated surface charge influences, and Gouy-Chapman theory provided a workable description of the ionic effects as arising from modulation of the pH at the surface of the RC. Incubation in D2O caused small increases in the pKs of the protonatable groups and a small, pH (pD)-dependent slowing of the binding rate. The salt, pH, temperature, viscosity, and D2O dependences of the proton uptake by RCs in the P+QA- state were accounted for by three considerations1) parallel pathways of H+ delivery to the RC, contributing to the observed (net) H+ disappearance; 2) rate limitation of the protonation of target groups within the protein by conformational dynamics; and 3) electrostatic influences of charged groups in the protein, via the surface pH.

译文

在特布瑞林存在下,通过阻断pH值和电导测定了球形球形红球菌R-26的分离反应中心(RC)的闪光诱导的H离子结合的动力学,并测定了叔丁烯的存在以阻止电子在伯醌和仲醌之间的转移( QA和QB),并且在没有外源电子给体到氧化的初级给体P的情况下,即P QA状态。在这些条件下,RC的质子结合是蛋白质而不是任何辅因子。光活化形成P QA-后,质子结合的动力学在所有研究的pH值下都是单指数的。在中性pH下,表观双分子速率常数接近质子在水溶液中的扩散极限(大约10(11)M-1 s-1),但在碱性pH范围内显着增加(例如2 x 10( 13)pH值为10的M-1 s-1。 pH依赖性的平均斜率是-0.4而不是-1.0,这可能是H扩散控制过程所期望的。高活化能(pH 8.0时为0.54 eV)和较弱的粘度依赖性表明RC吸收H离子不受扩散的限制。 H离子结合率与盐的依赖性以及反应中心质子化氨基酸残基的pK值都牵涉表面电荷影响,Gouy-Chapman理论提供了对由于在pH值下pH值的调节而产生的离子效应的可行描述。 RC的表面。在D2O中孵育会导致可质子化基团的pK小幅增加,并导致pH(pD)依赖性的结合速率减慢。 P QA状态下RC吸收质子的盐,pH,温度,粘度和D2O依赖性是由以下三个因素引起的: 2)通过构象动力学限制蛋白质内目标基的质子化速率;和3)通过表面pH值对蛋白质中带电基团的静电影响。

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