The redox potential of the Rieske [2Fe-2S] cluster of the bcl 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 Em = f312 f 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 14 up to Z = 2.5 M. From the cathodic-to-anodic peak separation, the heterogeneous rate constant, k", was calculated to be approximately 2 x 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 AS" = -155 J . K-' . mol-'. 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 pKa values of the oxidized protein, obtained by curve fitting, were 7.6 and 9.2, respectively. A group with a pK,,,, 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, center of the bcl complex.The ubiquitous bc complexes are constituents of the electron-transfer chains of mitochondria, chloroplasts and bacteria. All bc complexes contain two heme-b centers, one c-type heme (heme c1 or h e m e n and a Rieske iron/sulfur protein comprising a high-potential [2Fe-2S] cluster. The bcl complexes are embedded in the respective membranes. Cytochrome b forms the core of the complex, while cytochrome cf and the iron/sulfur protein have their redox centers within the aqueous domain and are linked to the membrane part of the complex through hydrophobic anchors.The Rieske iron/sulfur protein contains, like bacterial ferredoxins, a [2Fe-2S] cluster; however, its redox potential (+ 280 mV) is approximately 700 mV more positive than that of the ferredoxins (-420 mV). Evidence has been put forward that Rieske-type [2Fe-2S] clusters, which are also present in bacterial dioxygenases, have two histidine residues ligated to the Fez+ site, in contrast to the four cysteine ligation pattern of the ferredoxins [l -41.Determination of the redox potentials of the redox centers was essential for elucidating the electron transfer pathways of the bc complexes. These studies have been performed by spectropotentiometric titration of the hemes and by EPR monitored redox titration of the iron/sulfur cluster (for a review, see [5]). A disadvantage of this approach is the use of mediators which must not affect the measured potentials [6]. EPR of the iron/sulfur cluster is only possible in frozen samples at cryogenic temperature.Recently,...
A water-soluble fragment of the bc, complex from bovine heart mitochondria was isolated containing the intact Rieske [2Fe-2S] cluster. The fragment consists of the last 129 amino acid residues of the Rieske iron-sulfur protein and has a molecular mass of 14592 Da including two iron atoms. The absorption, visible CD, and EPR spectra of the fragment are indistinguishable from those of the membrane-bound iron-sulfur protein. The redox potential as determined by EPR-monitored redox titration was +306 mV. The far-ultraviolet CD spectrum is indicative of a protein with little regular secondary structure, while significant a-helix content was detected in the membrane anchor of the complete iron-sulfur protein. The fragment could be crystallized using poly(ethy1ene glycol) 6000 as precipitant. Needle-shaped single crystals have been grown by the hanging-drop vapor diffusion technique. These crystals belong to the space group P2, and diffract well beyond 0.2 nm resolution. Phase determination using the multiplewavelength anomalous-scattering technique is underway.
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