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°C and I of 0.01 M was E_m=+ 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 up to I= 2.5 M. From the cathodic‐to‐anodic peak separation, the heterogeneous rate constant, K°, was calculated to be approximately 2 × 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 S°=−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 pH 9, indicated two deprotonations of the oxidized protein. The pK_a values of the oxidized protein, obtained by curve fitting, were 7.6 and 9.2, respectively. A group with a pK_a,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 Q_o center of the bc₁ complex.

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