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

在特布林存在下,使用pH指示剂和电导法,测量了球形红球菌 (R-26菌株) 的分离反应中心 (RCs) 对闪光诱导的H + 离子结合的动力学,以阻断一级和二级醌之间的电子转移 (QA和QB),并且在没有外源电子供体的情况下,氧化的主供体P,即P QA态。在这些条件下,RCs的质子结合是与蛋白质结合,而不是与任何辅因子结合。光激活形成P QA-后,在所有研究的ph值下,质子结合的动力学都是单指数的。在中性pH下,表观双分子速率常数接近于水溶液中质子转移的扩散极限 (约10(11) M-1 s-1),但在碱性pH范围内显著增加 (例如,2 × 10(13) M-1在ph10 s-1)。pH依赖性的平均斜率为-0.4而不是-1.0,这对于H + 扩散控制的过程可能是预期的。高活化能 (pH 8.0时0.54 eV) 和弱粘度依赖性表明,RCs对H + 离子的吸收不受扩散的限制。H离子结合率的盐依赖性和反应中心的可质子化氨基酸残基的pk值涉及表面电荷的影响,Gouy-Chapman理论提供了对离子效应的可行描述,该离子效应是由pH的调节引起的。RC的表面。在D2O中孵育会导致可质子化基团的pKs小幅增加,并导致结合速率的pH (pD) 依赖性减慢。在P + QA状态下,RCs吸收质子的盐、酸碱度、温度、粘度和D2O依赖性由三个因素解释。1) H + 递送到RC的平行途径,有助于观察到的 (净) H + 消失; 2) 通过构象动力学限制蛋白质内目标基团质子化的速率; 3) 通过表面pH对蛋白质中带电基团的静电影响。

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