Baciou L, Rivas E, Sebban P
Laboratoire de Photosynthèse, Centre National de la Recherche Scientifique, Gif-sur-Yvette, France.
Biochemistry. 1990 Mar 27;29(12):2966-76. doi: 10.1021/bi00464a012.
The P+QA- and P+QB- charge recombination decay kinetics were studied in reaction centers from Rhodopseudomonas viridis reconstituted in phosphatidylcholine bilayer vesicles (proteoliposomes) and in chromatophores. P represents the primary electron donor, a dimer of bacteriochlorophyll; QA and QB are the primary and secondary stable quinone electron acceptors, respectively. In agreement with recent findings for reaction centers isolated in detergent [Sebban, P., & Wraight, C.A. (1989) Biochim. Biophys. Acta 974, 54-65] the P+QA- decay kinetics were biphasic (kfast and kslow). Arrhenius plots of the kinetics were linear, in agreement with the hypothesis of a thermally activated process (probably via P+I-; I is the first electron acceptor, a bacteriopheophytin) for the P+QA- charge recombination. Similar activation free energies (delta G) for this process were found in chromatophores and in proteoliposomes. Significant pH dependences of kfast and kslow were observed in chromtophores and in proteoliposomes. In the pH range 5.5-11, the pH titration curves of kfast and kslow were interpreted in terms of the existence of three protonable groups, situated between I- and QA-, which modulate the free energy difference between P+I- and P+QA-. In proteoliposomes, a marked effect of o-phenanthroline was observed on two of the three pKs, shifting one of them by more than 2 pH units. On the basis of recent structural data, we suggest a possible interpretation for this effect, which is much smaller in Rhodobacter sphaeroides. The decay kinetics of P+QB- were also biphasic. Marked pH dependences of the rate constants and of the relative proportions of both phases were also detected for these decays. The major conclusion of this work comes from the biphasicity of the P+QB- decay kinetics. We had suggested previously that biphasicity of the P+QA- charge recombination in Rps. viridis comes from nonequilibrium between protonation states of the reaction centers due to comparable rates of the protonation events and charge recombination. This hypothesis does not hold since the P+QB- decays occur on a time scale (tau approximately 300 ms at pH 8) much longer than protonation events. This leads to the conclusion that kfast and kslow (for both P+QA- and P+QB-) are related to conformational states of the reaction centers, existing before the flash. In addition, the fast and slow decays of P+QB- are related to those measured for P+QA-, via the calculations of the QA-QB in equilibrium QAQB- apparent equilibrium constants, K2.(ABSTRACT TRUNCATED AT 400 WORDS)
在磷脂酰胆碱双层囊泡（蛋白脂质体）和载色体中重构的绿脓杆菌反应中心，研究了P⁺QA⁻和P⁺QB⁻电荷复合衰变动力学。P代表初级电子供体，即细菌叶绿素二聚体；QA和QB分别是初级和次级稳定醌电子受体。与最近在去污剂中分离的反应中心的研究结果一致[Sebban, P., & Wraight, C.A. (1989) Biochim. Biophys. Acta 974, 54 - 65]，P⁺QA⁻衰变动力学是双相的（kfast和kslow）。动力学的阿伦尼乌斯图是线性的，这与P⁺QA⁻电荷复合的热激活过程（可能通过P⁺I⁻；I是第一个电子受体，细菌脱镁叶绿素）的假设一致。在载色体和蛋白脂质体中发现了该过程类似的活化自由能（ΔG）。在载色体和蛋白脂质体中观察到kfast和kslow对pH有显著依赖性。在pH范围5.5 - 11内，kfast和kslow的pH滴定曲线根据I⁻和QA⁻之间存在三个可质子化基团来解释，这些基团调节P⁺I⁻和P⁺QA⁻之间的自由能差。在蛋白脂质体中，邻菲罗啉对三个pK值中的两个有显著影响，使其中一个移动超过2个pH单位。根据最近的结构数据，我们对这种效应提出了一种可能的解释，这种效应在球形红细菌中要小得多。P⁺QB⁻的衰变动力学也是双相的。对于这些衰变，也检测到速率常数和两个相的相对比例对pH有显著依赖性。这项工作的主要结论来自P⁺QB⁻衰变动力学的双相性。我们之前曾提出，绿脓杆菌中P⁺QA⁻电荷复合的双相性源于反应中心质子化状态之间的非平衡，这是由于质子化事件和电荷复合的速率相当。这个假设不成立，因为P⁺QB⁻衰变发生的时间尺度（在pH 8时τ约为300毫秒）比质子化事件长得多。这导致得出结论，kfast和kslow（对于P⁺QA⁻和P⁺QB⁻两者）与闪光前存在的反应中心的构象状态有关。此外，通过计算QA - QB处于平衡状态QAQB⁻的表观平衡常数K2，P⁺QB⁻的快速和慢速衰变与P⁺QA⁻测量的衰变相关。
