Gray K A, Dutton P L, Daldal F
Department of Biology, Johnson Research Foundation, University of Pennsylvania, Philadelphia 19104.
Biochemistry. 1994 Jan 25;33(3):723-33. doi: 10.1021/bi00169a014.
Folding models suggest that the highly conserved histidine 217 of the cytochrome b subunit from the cytochrome bc1 complex is close to the quinone reductase (Qi) site. This histidine (bH217) in the cytochrome b polypeptide of the photosynthetic bacterium Rhodobacter capsulatus has been replaced with three other residues, aspartate (D), arginine (R), and leucine (L). bH217D and bH217R are able to grow photoheterotrophically and contain active cytochrome bc1 complexes (60% of wild-type activity), whereas the bH217L mutant is photosynthetically incompetent and contains a cytochrome bc1 complex that has only 10% of the wild-type activity. Single-turnover flash-activated electron transfer experiments show that cytochrome bH is reduced via the Qo site with near native rates in the mutant strains but that electron transfer between cytochrome bH and quinone bound at the Qi site is greatly slowed. These results are consistent with redox midpoint potential (Em) measurements of the cytochrome b subunit hemes and the Qi site quinone. The Em values of cyt bL and bH are approximately the same in the mutants and wild type, although the mutant strains have a larger relative concentration of what may be the high-potential form of cytochrome bH, called cytochrome b150. However, the redox properties of the semiquinone at the Qi site are altered significantly. The Qi site semiquinone stability constant of bH217R is 10 times higher than in the wild type, while in the other two strains (bH217D and bH217L) the stability constant is much lower than in the wild type. Thus H217 appears to have major effects on the redox properties of the quinone bound at the Qi site. These data are incorporated into a suggestion that H217 forms part of the binding pocket of the Qi site in a manner reminiscent of the interaction between quinone bound at the Qb site and H190 of the L subunit of the bacterial photosynthetic reaction center.
折叠模型表明，细胞色素bc1复合物中细胞色素b亚基高度保守的组氨酸217靠近醌还原酶（Qi）位点。光合细菌荚膜红细菌细胞色素b多肽中的这个组氨酸（bH217）已被另外三个残基取代，即天冬氨酸（D）、精氨酸（R）和亮氨酸（L）。bH217D和bH217R能够进行光异养生长，并含有活性细胞色素bc1复合物（为野生型活性的60%），而bH217L突变体则无光合能力，其细胞色素bc1复合物的活性仅为野生型的10%。单周转闪光激活电子转移实验表明，在突变菌株中，细胞色素bH通过Qo位点以接近天然的速率被还原，但细胞色素bH与结合在Qi位点的醌之间的电子转移大大减慢。这些结果与细胞色素b亚基血红素和Qi位点醌的氧化还原中点电位（Em）测量结果一致。在突变体和野生型中，细胞色素bL和bH的Em值大致相同，尽管突变菌株中可能是细胞色素bH的高电位形式（称为细胞色素b150）的相对浓度更高。然而，Qi位点半醌的氧化还原性质发生了显著改变。bH217R的Qi位点半醌稳定性常数比野生型高10倍，而在其他两个菌株（bH217D和bH217L）中，稳定性常数比野生型低得多。因此，H217似乎对结合在Qi位点的醌的氧化还原性质有重大影响。这些数据被纳入一个推测，即H217以类似于细菌光合反应中心Qb位点结合的醌与L亚基的H190之间相互作用的方式，构成Qi位点结合口袋的一部分。
