Ubiquinone pair in the Q(o) site central to the primary energy conversion reactions of cytochrome bc(1) complex
Ubiquinone pair in the Q(o) site central to the primary energy conversion reactions of cytochrome bc(1) complex
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DOI：
10.1021/bi00049a012
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发表时间：
1995-12-12
期刊：
影响因子：
2.9
通讯作者：
Dutton, PL
中科院分区：
文献类型：
--
作者：
Ding, H;Moser, CC;Dutton, PL
泛氢醌-细胞色素c氧化还原酶（cyt bet复合物）的核心机制是泛氢醌（QH（2））在Q（o）位点的催化氧化。QH（2）的氧化是由铁-cyt c引发的，由cyt c（1）和细胞色素bc（1）复合物的[2Fe-2S]簇介导。QH（2）氧化反过来驱动跨膜电子电荷分离，通过两个b型血红素在Q（i）位点处形成另一个泛醌（Q）。在早期的研究中，荚膜红细菌cytbc（1）复合物的含b-血红素多肽的残基F144和G158被证明对Q（o）位点功能有影响。在本研究中，F144和G158各自被中性残基单独取代，并且在强结合和弱结合Q（o）位点结构域（Q（os）和Q（ow））处测量Q和QH（2）的解离常数。发现F144或G158处的各种取代使Q和QH（2）在Q（os）和Q（ow）结构域的亲和力从零到超过10（3）倍减弱。这产生了一个Q（o）位点家族，其中Q（os）和Q（ow）结构域占据范围从几乎满到几乎空，在膜泛醌池（Q（pool））的普遍类似于3 × 10（-2）M浓度下。在每个突变体中，Q（os）结构域的亲和力通常保持比Q（ow）结构域的亲和力高10-20倍，如在野生型中所发现的，从而表明单个突变在每个结构域引起相当程度的弱化。此外，发现取代引起每个结构域中Q和QH（2）的亲和力的类似降低，从而将Q（o）位点的Q/QH（2）氧化还原中点电位（E（m7））保持在与野生型相似的值。通过对突变体家族中单次转换闪光产生的QH（2）氧化的产率和速率的测量表明，Q（os）和Q（ow）结构域在催化过程中发挥不同的作用。QH（2）氧化的产率与Q（os）结构域占有率（QH（2）或Q）线性相关，表明Q（os）结构域以比周转时间尺度慢得多的速率与Q（池）交换Q或QH（2）。另一方面，第一个QH（2）氧化的速率常数，在突变体中的范围从1620到1630。
The mechanistic heart of the ubihydroquinone-cytochrome c oxidoreductase (cyt bet complex) is the catalytic oxidation of ubihydroquinone (QH(2)) at the Q(o) site. QH(2) oxidation is initiated by ferri-cyt c, mediated by the cyt c(1) and [2Fe-2S] cluster of the cytochrome bc(1) complex. QH(2) oxidation in turn drives transmembrane electronic charge separation through two b-type hemes to another ubiquinone (Q) at the Q(i) site. In earlier studies, residues F144 and G158 of the b-heme containing polypeptide of the Rhodobacter capsulatus cyt bc(1) complex were shown to be influential in Q(o) site function. In the present study, F144 and G158 have each been singly substituted by neutral residues and the dissociation constants measured for both Q and QH(2) at each of the strong and weak binding Q(o) site domains (Q(os) and Q(ow)). Various substitutions at F144 or G158 were found to weaken the affinities for Q and QH(2) at both the Q(os) and Q(ow) domains variably from zero to beyond 10(3)-fold. This produced a family of Q(o) sites with Q(os) and Q(ow) domain occupancies ranging from nearly full to nearly empty at the prevailing similar to 3 x 10(-2) M concentration of the membrane ubiquinone pool (Q(pool)). In each mutant, the affinity of the Q(os) domain remained typically 10-20-fold higher than that of the Q(ow) domain, as is found for wild type, thereby indicating that the single mutations caused comparable extents of the weakening at each domain. Moreover, the substitutions were found to cause similar decreases of the affinities of both Q and QH(2) in each domain, thereby maintaining the Q/QH(2) redox midpoint potentials (E(m7)) of the Q(o) site at values similar to that of the wild type. Measurement of the yield and rate of QH(2) oxidation generated by single turnover flashes in the family of mutants suggests that the Q(os) and Q(ow) domains serve different roles for the catalytic process. The yield of the QH(2) oxidation correlates linearly with Q(os) domain occupancy (QH(2) or Q), suggesting that the Q(os) domain exchanges Q or QH(2) with the Q(pool) at a rate which is much slower than the time scale of turnover. On the other hand, the rate constants of the first QH(2) oxidation, ranging in the mutants from 1620 to