DETERMINATION OF THE REDOX PROPERTIES OF THE RIESKE [2FE-2S] CLUSTER OF BOVINE HEART BC1 COMPLEX BY DIRECT ELECTROCHEMISTRY OF A WATER-SOLUBLE FRAGMENT

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 quasi-reversible response. The midpoint potential at pH 7.2, 25-degrees-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 square-root 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 DELTAS-degrees = -155J . 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.