CD‐monitored redox titration of the Rieske Fe‐S protein of Rhodobacter sphaeroides: pH dependence of the midpoint potential in isolated bc 1 complex and in membranes

摘要

>The redox potential of the Rieske Fe-S protein has been investigated using circular dichroism (CD)-spectroscopy. The CD features characteristic of the purified bc ₁ complex and membranes of Rhodobacter sphaeroides were found in the region between 450 and 550 nm. The difference between reduced and oxidized CD-spectra shows a negative band at about 500 nm with a half of width 30 nm that corresponds to the specific dichroic absorption of the reduced Rieske protein (Fee, J.A. et al. (1984) J. Biol. Chem. 259, 124–133; Degli Esposti, M. et al. (1987) Biochem. J. 241, 285–290; Rich, P.R. and Wiggins, T.E. (1992) Biochem. Soc. Trans. 20, 241S). It was found that the redox potential at pH 7.0 for the Rieske center in the isolated bc ₁ complex and in chromatophore membranes from the R-26 strain of Rb. sphaeroides is 300±5 mV. In chromatophores from the BC17C strain of Rb. sphaeroides, the E _m value measured for the Rieske iron-sulfur protein (ISP) was higher (315±5 mV), but the presence of carotenoids made measurement less accurate. The E _m varied with pH in the range above pH 7, and the pH dependence was well fit either by one pK at ∼7.5 in the range of titration, or by two pK values, pK ₁=7.6 and pK ₂=9.8. Similar titrations and pK values were found for the Rieske Fe-S protein in the isolated bc ₁ complex and membranes from the R-26 strain of Rb. sphaeroides. The results are discussed in the context of the mechanism of quinol oxidation by the bc ₁ complex, and the role of the iron sulfur protein in formation of a reaction complex at the Q_o-site.