Gerencsér László, Maróti Péter
Department of Biophysics, University of Szeged, Egyetem utca 2, Szeged, Hungary H-6722.
Biochemistry. 2006 May 2;45(17):5650-62. doi: 10.1021/bi052071m.
Photosynthetic reaction centers produce and export oxidizing and reducing equivalents in expense of absorbed light energy. The formation of fully reduced quinone (quinol) requires a strict (1:1) stoichiometric ratio between the electrons and H(+) ions entering the protein. The steady-state rates of both transports were measured separately under continuous illumination in the reaction center from the photosynthetic bacterium Rhodobacter sphaeroides. The uptake of the first proton was retarded by different methods and made the rate-limiting reaction in the photocycle. As expected, the rate constant of the observed proton binding remained constant (7 s(-)(1)), but that of the cytochrome photooxidation did show a remarkably large increase from 14 to 136 s(-)(1) upon increase of the exciting light intensity up to 5 W/cm(2) (808 nm) at pH 8.4 in the presence of NiCl(2). This corresponds to about 20:1 (e(-):H(+)) stoichiometric ratio. The observed enhancement is linearly proportional to the light intensity and the rate constant of the proton uptake by the acceptor complex and shows saturation character with quinone availability. For interpretation of the acceleration of cytochrome turnover, an extended model of the photocycle is proposed. A fraction of photochemically trapped RC can undergo fast (>10(3) s(-)(1)) conformational change where the semiquinone loses its high binding affinity (the dissociation constant increases by more than 5 orders of magnitude) and dissociates from the Q(B) binding site of the protein with a high rate of 4000 s(-)(1). Concomitantly, superoxide is being produced. No H(+) ion is taken up, and no quinol is created by the photocycle which is operating in about 25% of the reaction centers at the highest light intensity (5500 s(-)(1)) and slowest proton uptake (3.5 s(-)(1)) used in our experiments. The possible physical background of the light-induced conformational change and the relationship between the energies of dissociation and redox changes of the quinone in the Q(B) binding sites are discussed.
光合反应中心以吸收的光能为代价产生并输出氧化还原当量。完全还原醌（醌醇）的形成需要进入蛋白质的电子与氢离子之间严格的（1:1）化学计量比。在来自光合细菌球形红杆菌的反应中心连续光照下，分别测量了两种运输的稳态速率。通过不同方法延迟了第一个质子的摄取，使其成为光循环中的限速反应。正如预期的那样，观察到的质子结合速率常数保持恒定（7 s⁻¹），但在pH 8.4、存在NiCl₂的情况下，当激发光强度增加到5 W/cm²（808 nm）时，细胞色素光氧化的速率常数确实从14显著增加到136 s⁻¹。这对应于约20:1（e⁻:H⁺）的化学计量比。观察到的增强与光强度以及受体复合物摄取质子的速率常数呈线性比例关系，并表现出对醌可用性的饱和特性。为了解释细胞色素周转的加速，提出了一个扩展的光循环模型。一部分光化学捕获的反应中心（RC）可以经历快速（>10³ s⁻¹）的构象变化，其中半醌失去其高结合亲和力（解离常数增加超过5个数量级）并以4000 s⁻¹的高速从蛋白质的Q(B)结合位点解离。同时，会产生超氧化物。在我们实验中使用的最高光强度（5500 s⁻¹）和最慢质子摄取（3.5 s⁻¹）下，约25%的反应中心中运行的光循环不摄取H⁺离子，也不产生醌醇。讨论了光诱导构象变化的可能物理背景以及Q(B)结合位点中醌的解离能与氧化还原变化之间的关系。
