A spectroscopic method for observing the domain movement of the Rieske iron–sulfur protein

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...

On the Spatial Organization of Hemes and Chlorophyll in Cytochrome b 6 f

“…In bc 1 crystals, the movement of the Rieske protein is not accompanied by a significant displacement of Cyt c 1 . However, recent EPR data indicate that the conformational change of the Rieske protein in both the bc 1 and b 6 f complexes can also be induced by modulating its redox state (68). There are, to our knowledge, no data yet regarding the effect of redox potential on heme orientation in either the bc 1 or the b 6 f complex.…”

Relative Orientation of the Hemes of the Cytochromebc 1 Complexes from Rhodobacter sphaeroides, Rhodospirillum rubrum, and Beef Heart Mitochondria

“…One must remember that our LD study was performed in the reduced state, whereas the crystallographic structure published by Zhang et (12). Moreover, the relative orientation of b-hemes has been shown to be highly dependent of the presence of specific inhibitors in the Q i and/or Q o sites in the case of the chicken heart mitochondrial complex by Zhang et al (5).…”

“…This would require the crystallographic analysis of the complex in the reduced state. Alternatively, the comparison of LD signals of oriented samples chemically reduced to oriented samples reduced at low temperature (and therefore in a frozen immobilized oxidized state) by ␥-ray ionization as recently described (12) can bring new insights on this proposal. These experiments are under progress.…”

Protonmotive pathways and mechanisms in the cytochrome bc1 complex

“…Molecular dynamics simulation predicted a steered but stochastic movement [15]. In contrast, electron paramagnetic resonance studies of oriented membranes suggest a redox dependent movement of the domain [16]. The true nature of the movement remains to be elucidated.…”