Baciou L, Bylina E J, Sebban P
UPR 407, Bat. 24, Centre National de le Recherche Scientifique Gif/Yvette, France.
Biophys J. 1993 Aug;65(2):652-60. doi: 10.1016/S0006-3495(93)81114-7.
Reaction centers from the purple bacterium Rhodobacter (Rb.) capsulatus and from two mutants ThrL226-->Ala and IleL229-->Ser, modified in the binding protein pocket of the secondary quinone acceptor (QB), have been studied by flash-induced absorbance spectroscopy. In ThrL226-->Ala, the binding affinities for endogenous QB (ubiquinone 10) and UQ6 are found to be two to three times as high as the wild type. In contrast, in IleL229-->Ser, the binding affinity for UQ6 is decreased about three times compared to the wild type. In ThrL226-->Ala, a markedly increased sensitivity (approximately 30 times) to o-phenanthroline is observed. In Rhodopseudomonas viridis, where Ala is naturally in position L226, the sensitivity to o-phenanthroline is close to that observed in ThrL226-->Ala. We propose that the presence of Ala in position L226 is responsible for the high sensitivity to that inhibitor. The pH dependencies of the rate constants of P+QB- (kBP) charge recombination kinetics (P is a dimer of bacteriochlorophyll, and QB is the secondary quinone electron acceptor) show destabilization of QB- in ThrL226-->Ala and IleL229-->Ser, compared to the wild type. At low pH, similar apparent pK values of protonation of amino acids around QB- are measured in the wild type and the mutants. In contrast to Rb. sphaeroides, in the wild type Rb. capsulatus, kBP substantially increases in the pH range 7-10. This may reflect some differences in the respective structures of both strains or, alternatively, may be due to deprotonation of TyrL 215 in Rb. capsulatus. At pH 7, measurements of the rate constant of QA to QB electron transfer reveal a threefold greater rate in the reaction centers from wild type Rb. capsulatus (65 +/- 1 0 ps)-1 compared to Rb. sphaeroides.We suggest that this may arise from a 0.7-A smaller distance between the quinones in the former strain. Our spectroscopic data on the wild type Rb. capsulatus reaction center suggest the existence of notable differences with the Rb. sphaeroides reaction center structure.
利用闪光诱导吸收光谱法，对来自荚膜红细菌（Rb.）的反应中心以及在次级醌受体（QB）的结合蛋白口袋中发生ThrL226→Ala和IleL229→Ser突变的两个突变体的反应中心进行了研究。在ThrL226→Ala突变体中，对内源QB（泛醌10）和UQ6的结合亲和力是野生型的两到三倍。相比之下，在IleL229→Ser突变体中，对UQ6的结合亲和力相较于野生型降低了约三倍。在ThrL226→Ala突变体中，观察到对邻菲罗啉的敏感性显著增加（约30倍）。在绿假单胞菌中，L226位点天然存在丙氨酸，其对邻菲罗啉的敏感性与在ThrL226→Ala突变体中观察到的相近。我们认为L226位点存在丙氨酸是对该抑制剂具有高敏感性的原因。P + QB- （kBP）电荷复合动力学（P是细菌叶绿素二聚体，QB是次级醌电子受体）的速率常数的pH依赖性表明，与野生型相比，ThrL226→Ala和IleL229→Ser突变体中QB- 的稳定性降低。在低pH值下，野生型和突变体中QB- 周围氨基酸质子化的表观pK值相似。与球形红细菌不同，在野生型荚膜红细菌中，kBP在pH范围7 - 10内大幅增加。这可能反映了两种菌株各自结构上的一些差异，或者可能是由于荚膜红细菌中TyrL 215的去质子化。在pH 7时，QA到QB电子转移速率常数的测量结果显示，野生型荚膜红细菌反应中心的反应速率（65 ± 10 ps）-1比球形红细菌高三倍。我们认为这可能是由于前一种菌株中醌之间的距离小0.7 Å。我们关于野生型荚膜红细菌反应中心的光谱数据表明，其与球形红细菌反应中心结构存在显著差异。
