The PufX membrane protein is essential for photosynthetic growth of Rhodobacter sphaeroides because it is required for multiple-turnover electron transfer under anaerobic conditions [see accompanying article; Barz, W. P., Francia, F., Venturoli, G., Melandri, B. A., Verméglio, A., & Oesterhelt, D. (1995) Biochemistry 34, 15235-15247]. In order to understand the molecular role of PufX, light-induced absorption spectroscopy was performed using a pufX- mutant, a pufX+ strain, and two suppressor mutants. We show that the reaction center (RC) requires PufX for its functionality under different redox conditions than the cytochrome bc1 complex: When the kinetics of flash-induced reduction of cytochrome b561 were monitored in chromatophores, we observed a requirement of PufX for turnover of the cytochrome bc1 complex only at high redox potential (Eh > 140 mV), suggesting a function of PufX in lateral ubiquinol transfer from the RC. In contrast, PufX is required for multiple turnover of the RC only under reducing conditions: When the Q pool was partially oxidized in vivo using oxygen or electron acceptors like dimethyl sulfoxide or trimethylamine N-oxide, the deletion of PufX had no effect on light-driven electron flow through the RC. Flash train experiments under anaerobic in vivo conditions revealed that RC photochemistry does not depend on PufX for the first two flash excitations. Following the third and subsequent flashes, however, efficient charge separation requires PufX, indicating an important role of PufX for fast Q/QH2 exchange at the QB site of the RC. We show that the Q/QH2 exchange rate is reduced approximately 500-fold by the deletion of PufX when the Q pool is nearly completely reduced, demonstrating an essential role of PufX for the access of ubiquinone to the QB site. The fast ubiquinone/ubiquinol exchange is partially restored by suppressor mutations altering the macromolecular antenna structure. These results suggest an indirect role of PufX in structurally organizing a functional photosynthetic apparatus.

译文

PufX膜蛋白对于球形红球菌的光合生长至关重要,因为它是厌氧条件下多转换电子转移所必需的 [见随附文章; Barz,W. P.,Francia,F.,Venturoli,G.,Melandri,B. A.,verm é glio,A.,& Oesterhelt,D. (1995) 生物化学34,15235-15247]。为了了解PufX的分子作用,使用pufX突变体,pufX菌株和两个抑制突变体进行了光诱导吸收光谱。我们表明,反应中心 (RC) 需要PufX在与细胞色素bc1复合物不同的氧化还原条件下的功能: 当在色谱中监测快速诱导的细胞色素b561还原的动力学时,我们观察到仅在高氧化还原电位 (Eh > 140 mV) 下对细胞色素bc1复合物的周转需要PufX,这表明PufX在从RC的侧向泛醇转移中的功能。相反,仅在还原条件下,才需要PufX进行RC的多次周转: 当使用氧或电子受体 (如二甲基亚砜或三甲胺N-氧化物) 在体内部分氧化Q池时,PufX的缺失对通过RC的光驱动电子流没有影响。在厌氧体内条件下进行的闪光训练实验表明,前两个闪光激发的RC光化学不依赖于PufX。然而,在第三次及随后的闪烁之后,有效的电荷分离需要PufX,这表明PufX对于RC的QB站点的快速Q/QH2交换具有重要作用。我们表明,当Q池几乎完全减少时,通过PufX的删除,Q/QH2汇率降低了大约500倍,这表明PufX对于泛醌进入QB站点至关重要。通过改变大分子天线结构的抑制突变,部分恢复了快速的泛醌/泛醌交换。这些结果表明,PufX在结构上组织功能性光合作用装置方面具有间接作用。

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