Determination of the redox properties of the Rieske [2Fe‐2S] cluster of bovine heart bc₁ complex by direct electrochemistry of a water‐soluble fragment

Abstract

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,...

Electrochemical study of the redox properties of [2Fe‐2S] ferredoxins Evidence for superreduction of the Rieske [2Fe‐2S] cluster

“…The independence of pH is consistent with the postulated coordination of these type of clusters. As described in earlier work the I 400 nA pH dependence of the first transition can be explained by the protonation of histidines coordinated to the one of the iron atoms in the cluster [6]. However the other Fe-atom is coordinated by cysteines.…”

“…Cyclic voltammetry of the water soluble fragment of the Rieske protein in the range of -100 mV to 700 mV versus SHE results in well defined voltammograms as described in ref [6]. Upon extension of the scan range to lower potentials a second redox transition was observed at -840 mV vs. SHE.…”

“…The water-soluble fragment of the Rieske protein of bovine heart bc l complex was isolated and purified as described previously [5,6]. Ferredoxin:NADP + oxidoreductase from spinach leaves and [2Fe-2S] ferredoxin from Spiru/ina platensis were obtained from Sigma Chemie (Bornem, Belgium) as the salt-free lyophilizate.…”

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what determines the reduction potential(s) of these proteins, and what determines whether their functioning is associated with the transfer of no, one, one pair, or multiple pairs of electrons.By a fortunate coincidence of circumstances we have found it possible to study the complete redox behaviour of a biological [2Fe-2S] system: (i) the Rieske protein can be purified as a water-soluble fragment with no observable change in its ironsulfur cluster [5]; (ii) the fragment exhibits a direct, unmediated electrochemical response on glassy carbon [6]; (iii) the first reduction potential of the Rieske cluster is unusually high, namely Era,7 = + 0.3 V [7]; (iv) carbon electrodes have a high overpotential for H 2 evolution, therefore, their use allows for aqueous-solution bio-electrochemistry down to E = -1 V [8]. The voltammetric characterization of the Rieske cluster and the comparison of its properties with other (i.e.

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Ironies in Photosynthetic Electron Transport: A Personal Perspective

“…Following a protocol similar to that of Thomas Link, used for purification of an analogous soluble fragment from the Cyt bc 1 complex (Link et al 1992), Huamin used an exogenous protease, thermolysin, to cleave an N-terminal 40-residue segment from the Rieske protein in the complex. Although there are differences in the p-side surface of the mitochondrial Cyt bc 1 complex, and the Cyt b 6 f complex from spinach, this strategy of cutting off at its roots the peripheral domain of an embedded membrane protein worked for both complexes.…”