Robertson D E, Prince R C, Bowyer J R, Matsuura K, Dutton P L, Ohnishi T
J Biol Chem. 1984 Feb 10;259(3):1758-63.
The antimycin-sensitive ubisemiquinone radical (QC) of the ubiquinol-cytochrome c oxidoreductase of submitochondrial particles and chromatophores of Rhodopseudomonas sphaeroides Ga has been studied by a combination of redox potentiometry and EPR spectroscopy. This g = 2.005 radical signal appears at physiological pH values and increases in intensity with increasing pH up to pH 7.6 in submitochondrial particles and pH 9.0 in R. sphaeroides after which its intensity remains unchanged. The Em7 (ubiquinone/quinol) of the signal, estimated from redox titration data is 80 mV for submitochondrial particles, and 150 mV in chromatophores. Each of these values is higher than that of the quinone pool by 20 mV in submitochondrial particles and 60 mV in R. sphaeroides. This indicates that the quinone at the binding site is out of equilibrium with the pool, and that binding site preferentially binds quinol over quinone. Analysis of the shapes of the semiquinone titration curves, taken together with the midpoint elevation, indicates a quinone-binding site: cytochrome c1 stoichiometry of 1:1 in both submitochondrial particles and chromatophores. At its maximal intensity, the semiquinone concentration at the binding site is 0.26 in submitochondrial particles (greater than pH 7.6) and 0.4 in chromatophores (greater than pH 9.0). In both systems, the midpoint of the ubiquinone/ubisemiquinone couple is constant as the pH is raised up to the pH of maximal semiquinone formation whereafter it becomes more negative at the rate of -60 mV/pH unit. The midpoint of the ubisemiquinone/quinol couple, on the other hand, varies by -120 mV/pH unit at pH values up to the transition pH, after which it, too, changes by -60 mV/pH unit. This seemingly anomalous behavior may be explained by invoking a protonated group at or near the quinone-binding site whose pK corresponds to the pH transition point in the quinone/semiquinone/quinol redox chemistry when the site is free or when quinone or quinol occupies the site. This pK is elevated to at least pH 9.0 in submitochondrial particles and 10.5 in R. sphaeroides when semiquinone is bound to the site.
通过氧化还原电位测定法和电子顺磁共振光谱法相结合，对球形红假单胞菌Ga亚线粒体颗粒和载色体中泛醇 - 细胞色素c氧化还原酶的抗霉素敏感泛半醌自由基（QC）进行了研究。这个g = 2.005的自由基信号在生理pH值下出现，在亚线粒体颗粒中，其强度随着pH值升高至7.6而增加，在球形红假单胞菌中pH值升高至9.0时强度增加，之后强度保持不变。根据氧化还原滴定数据估算，该信号的Em7（泛醌/泛醇）在亚线粒体颗粒中为80 mV，在载色体中为150 mV。在亚线粒体颗粒中，这些值中的每一个都比醌池的值高20 mV，在球形红假单胞菌中高60 mV。这表明结合位点处的醌与醌池处于非平衡状态，并且结合位点优先结合泛醇而非醌。对半醌滴定曲线形状的分析，结合中点升高情况，表明在亚线粒体颗粒和载色体中醌结合位点与细胞色素c1的化学计量比均为1:1。在其最大强度时，结合位点处的半醌浓度在亚线粒体颗粒中（pH大于7.6）为0.26，在载色体中（pH大于9.0）为0.4。在这两个系统中，泛醌/泛半醌偶联的中点在pH升高至半醌形成最大时保持恒定，此后以-60 mV/pH单位的速率变得更负。另一方面，泛半醌/泛醇偶联的中点在pH值达到转变pH之前以-120 mV/pH单位变化，之后也以-60 mV/pH单位变化。这种看似异常的行为可以通过在醌结合位点或其附近存在一个质子化基团来解释，其pK对应于当该位点游离或醌或泛醇占据该位点时醌/半醌/泛醇氧化还原化学中的pH转变点。当半醌结合到该位点时，这个pK在亚线粒体颗粒中升高到至少pH 9.0，在球形红假单胞菌中升高到10.5。
