No abstract

The g-tensor orientation of the chemically reduced Rieske cluster in cytochrome bc1 complex from Rhodovulum sulfidophilum with respect to the membrane was determined in the presence and absence of inhibitors and in the presence of oxidized and reduced quinone in the quinol-oxidizing-site (Qo-site) by EPR on twodimensionally ordered samples. Almost identical orientations were observed when oxidized or reduced quinone, stigmatellin, or 5-(n-undecyl)-6-hydroxy-4,7-dioxobenzothiazole was present. Occupancy of the Qo-site by myxothiazole induced appearance of a minority population with a substantially differing conformation and presence of E-␤-methoxyacrylate-stilbene significantly reduced the contribution of the major conformation observed in the other cases. Furthermore, when the oxidized iron-sulfur cluster was reduced at cryogenic temperatures by the products of radiolysis, the orientation of its magnetic axes was found to differ significantly from that of the chemically reduced center. The ''irradiation-induced'' conformation converts to that of the chemically reduced center after thawing of the sample. These results confirm the effects of Qo-site inhibitors on the equilibrium conformation of the Rieske iron-sulfur protein and provide evidence for a reversible redox-influenced interconversion between conformational states. Moreover, the data obtained with the iron-sulfur protein demonstrate that the conformation of ''EPR-inaccessible'' reduction states of redox centers can be studied by inducing changes of redox state at cryogenic temperatures. This technique appears applicable to a wide range of comparable electron transfer systems performing redox-induced conformational changes. T he cytochrome bc-complex is the only energy-coupling membrane-integral enzyme common to both photosynthetic and respiratory electron transport chains. In addition to three heme groups, the enzyme contains an unusual [2Fe2S] cluster, the so-called Rieske center. A key enzymatic feature of the complex is the bifurcation of the two electrons derived from oxidation of a quinol molecule at a catalytic site (the ''Q o -site'') into two distinct electron transfer chains within the complex. The Q o -site is positioned between a b-type heme and the [2Fe2S]-cluster of the Rieske iron-sulfur protein (ISP), and the ISP protein has been shown to play a decisive role in turnover at the site (1). Apart from the physiological quinone, the Q o -site binds several competitive and noncompetitive inhibitors of catalysis (mostly quinone analogs) such as stigmatellin, 5-(n-undecyl)-6-hydroxy-4,7-dioxobenzothiazole (UHDBT), myxothiazole, or E-␤-methoxyacrylate (MOA) stilbene.In the recent x-ray structure analyses of the mitochondrial cytochrome bc 1 complex (2-5), the Rieske protein was found in three distinct positions with respect to the remaining subunits of the enzyme, i.e., in a geometry positioning the of the enzyme during turnover, and a domain movement of the Rieske protein was implied as a prerequisite for efficient electron transfer (3-5). A deta...

Herbicides of the triazine class block electron transfer in the photosynthetic reaction centers of purple bacteria and PSII of higher plants. They are thought to act by competing with one of the electron acceptors, the secondary quinone, QB, for its binding site. Several mutants of the purple bacterium Rhodopseudomonas viridis resistant to terbutryn [2-(methylthio)-4-(ethylamino)-6-(tert-butylamino)-s-triazine] have been isolated by their ability to grow photosynthetically in the presence of the herbicide. Sequence analysis of the genes coding for the L and M subunits of the reaction center showed that four different mutants were obtained, two of them being double mutated: T1 (SerL223----Ala and ArgL217----His), T3 (PheL216----Ser and ValM263----Phe), T4 (TyrL222----Phe), and T6 (PheL216----Ser). The residues L223 and L216 are involved in binding of QB, whereas L217 and L222 are not. M263 is part of the binding pocket of the primary quinone, QA. The affinity of the reaction centers for terbutryn and the electron transfer inhibitor o-phenanthroline, determined via the biphasic charge recombination after one flash, is decreased for all mutants. The affinity for ubiquinone 9 is also decreased, except in T1. Characterization by EPR spectroscopy showed that the QB.-Fe2+ signal of T4, having a g = 1.93 peak, is different from the signals obtained with the wild type and the other mutants but very similar to those of Rhodospirillum rubrum and PSII. The results obtained by the combination of these different techniques are discussed with respect to the three-dimensional structure of the wild type and the mode of binding of ubiquinone, terbutryn, and o-phenanthroline as determined by X-ray structure analysis.