Meinhardt S W, Yang X H, Trumpower B L, Ohnishi T
J Biol Chem. 1987 Jun 25;262(18):8702-6.
The ubiquinol-cytochrome c oxidoreductase (cytochrome bc1) complex from Paracoccus denitrificans exhibits a thermodynamically stable ubisemiquinone radical detectable by EPR spectroscopy. The radical is centered at g = 2.004, is sensitive to antimycin, and has a midpoint potential at pH 8.5 of +42 mV. These properties are very similar to those of the stable ubisemiquinone (Qi) previously characterized in the cytochrome bc1 complexes of mitochondria. The micro-environment of the Rieske iron-sulfur cluster in the Paracoccus cytochrome bc1 complex changes in parallel with the redox state of the ubiquinone pool. This change is manifested as shifts in the gx, gy, and gz values of the iron-sulfur cluster EPR signal from 1.80, 1.89, and 2.02 to 1.76, 1.90, and 2.03, respectively, as ubiquinone is reduced to ubiquinol. The spectral shift is accompanied by a broadening of the signal and follows a two electron reduction curve, with a midpoint potential at pH 8.5 of +30 mV. A hydroxy analogue of ubiquinone, UHDBT, which inhibits respiration in the cytochrome bc1 complex, shifts the gx, gy, and gz values of the iron-sulfur cluster EPR signal to 1.78, 1.89, and 2.03, respectively, and raises the midpoint potential of the iron-sulfur cluster at pH 7.5 from +265 to +320 mV. These changes in the micro-environment of the Paracoccus Rieske iron-sulfur cluster are like those elicited in mitochondria. These results indicate that the cytochrome bc1 complex of P. denitrificans has a binding site for ubisemiquinone and that this site confers properties on the bound ubisemiquinone similar to those in mitochondria. In addition, the line shape of the Rieske iron-sulfur cluster changes in response to the oxidation-reduction status of ubiquinone, and the midpoint of the iron-sulfur cluster increases in the presence of a hydroxyquinone analogue of ubiquinone. The latter results are also similar to those observed in the mitochondrial cytochrome bc1 complex. However, unlike the mitochondrial complexes, which contain eight to 11 polypeptides and are thought to contain distinct quinone binding proteins, the Paracoccus cytochrome bc1 complex contains only three polypeptide subunits, cytochromes b, c1, and iron-sulfur protein. The ubisemiquinone binding site and the site at which ubiquinone and/or ubiquinol bind to affect the Rieske iron-sulfur cluster in Paracoccus thus exist in the absence of any distinct quinone binding proteins and must be composed of domains contributed by the cytochromes and/or iron-sulfur protein.
反硝化副球菌的泛醇 - 细胞色素c氧化还原酶（细胞色素bc1）复合物表现出一种可用电子顺磁共振波谱检测到的热力学稳定的泛半醌自由基。该自由基的g值为2.004，对抗霉素敏感，在pH 8.5时的中点电位为 +42 mV。这些性质与先前在线粒体细胞色素bc1复合物中表征的稳定泛半醌（Qi）的性质非常相似。反硝化副球菌细胞色素bc1复合物中 Rieske 铁硫簇的微环境随泛醌池的氧化还原状态平行变化。这种变化表现为随着泛醌还原为泛醇，铁硫簇电子顺磁共振信号的gx、gy和gz值分别从1.80、1.89和2.02移至1.76、1.90和2.03。光谱位移伴随着信号变宽，并遵循双电子还原曲线，在pH 8.5时的中点电位为 +30 mV。泛醌的羟基类似物UHDBT可抑制细胞色素bc1复合物中的呼吸作用，它将铁硫簇电子顺磁共振信号的gx、gy和gz值分别移至1.78、1.89和2.03，并将铁硫簇在pH 7.5时的中点电位从 +265 mV提高到 +320 mV。反硝化副球菌Rieske铁硫簇微环境的这些变化与线粒体中引发的变化相似。这些结果表明，反硝化副球菌的细胞色素bc1复合物具有一个泛半醌结合位点，并且该位点赋予结合的泛半醌与线粒体中相似的性质。此外，Rieske铁硫簇的线形响应泛醌的氧化还原状态而变化，并且在泛醌的羟基醌类似物存在下铁硫簇的中点电位升高。后一个结果也与在线粒体细胞色素bc1复合物中观察到的结果相似。然而，与含有八到十一种多肽且被认为含有不同醌结合蛋白的线粒体复合物不同，反硝化副球菌细胞色素bc1复合物仅包含三个多肽亚基，即细胞色素b、c1和铁硫蛋白。因此，反硝化副球菌中泛半醌结合位点以及泛醌和/或泛醇结合以影响Rieske铁硫簇的位点存在于没有任何不同醌结合蛋白的情况下，并且必须由细胞色素和/或铁硫蛋白贡献的结构域组成。
