Link T A, Hagen W R, Pierik A J, Assmann C, von Jagow G
Universitätsklinikum Frankfurt, Zentrum für Biologische Chemie, Therapeutische Biochemie, Federal Republic of Germany.
Eur J Biochem. 1992 Sep 15;208(3):685-91. doi: 10.1111/j.1432-1033.1992.tb17235.x.
The redox potential of the Rieske [2Fe-2S] cluster of the bc1 complex from bovine heart mitochondria was determined by cyclic voltammetry of a water-soluble fragment of the iron/sulfur protein. At the nitric-acid-treated bare glassy-carbon electrode, the fragment gave an immediate and stable quasireversible response. The midpoint potential at pH 7.2, 25 degrees C and I of 0.01 M was Em = +312 +/- 3 mV. This value corresponds within 20 mV to results of an EPR-monitored dye-mediated redox titration. With increasing ionic strength, the midpoint potential decreased linearly with square root of I up to I = 2.5 M. From the cathodic-to-anodic peak separation, the heterogeneous rate constant, k degrees, was calculated to be approximately 2 x 10(-3) cm/s at low ionic strength; the rate constant increased with increasing ionic strength. From the temperature dependence of the midpoint potential, the standard reaction entropy was calculated as delta S degrees = -155 J.K-1.mol-1. The pH dependence of the midpoint potential was followed over pH 5.5-10. Above pH 7, redox-state-dependent pK changes were observed. The slope of the curve, -120 mV/pH above pH9, indicated two deprotonations of the oxidized protein. The pKa values of the oxidized protein, obtained by curve fitting, were 7.6 and 9.2, respectively. A group with a pKa,ox of approximately 7.5 could also be observed in the optical spectrum of the oxidized protein. Redox-dependent pK values of the iron/sulfur protein are considered to be essential for semiquinone oxidation at the Qo center of the bc1 complex.
通过对铁硫蛋白的水溶性片段进行循环伏安法，测定了牛心线粒体bc1复合物中 Rieske [2Fe-2S] 簇的氧化还原电位。在硝酸处理过的裸玻碳电极上，该片段给出了即时且稳定的准可逆响应。在pH 7.2、25℃和离子强度I为0.01 M时，中点电位为Em = +312±3 mV。该值与EPR监测的染料介导的氧化还原滴定结果在20 mV范围内相符。随着离子强度增加，中点电位随I的平方根线性下降，直至I = 2.5 M。根据阴极到阳极的峰间距，在低离子强度下计算出异相速率常数k°约为2×10⁻³ cm/s；速率常数随离子强度增加而增大。根据中点电位的温度依赖性，计算出标准反应熵为ΔS° = -155 J·K⁻¹·mol⁻¹。在pH 5.5 - 10范围内跟踪中点电位的pH依赖性。在pH 7以上，观察到氧化还原状态依赖性的pK变化。pH 9以上曲线的斜率为-120 mV/pH，表明氧化态蛋白有两次去质子化。通过曲线拟合得到的氧化态蛋白的pKa值分别为7.6和9.2。在氧化态蛋白的光谱中也可观察到pKa,ox约为7.5的基团。铁硫蛋白的氧化还原依赖性pK值被认为对于bc1复合物Qo中心的半醌氧化至关重要。
