The recently detected Rieske iron - sulfur center in the membrane of the thermoacidophilic archaeon Sulfolobus acidocaldarius (Anemuller et al., 1993, FEBS Lett. 318, 61 - 64) was further characterized by EPR spectroscopy, coupled to redox potentiometry and functional studies. The reduction potential is pH - dependent above pH 6, revealing the influence of two ionization equilibria in the oxidized form, with pK(a)(ox) - values of 6.2 and 8.5. Above pH 9, the slope of the curve is - 120 mV/pH - unit. A partially purified fraction exerted a ubiquinol - cytochrome c oxidoreductase activity. To our knowledge, for the first time, in a membrane bound Rieske iron - sulfur protein, unequivocal evidence for a two proton dependent redox equilibrium is presented. (C) 1994 Academic Press, Inc.