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

The g-tensor orientation of the chemically reduced Rieske cluster in cytoch rome bc(1) complex from Rhodovolum sulfidophilum with respect to the membra ne was determined in the presence and absence of inhibitors and in the pres ence of oxidized and reduced quinone in the quinol-oxidizing-site (Q(o)-sit e) by EPR on two-dimensionally ordered samples, Almost identical orientatio ns were observed when oxidized or reduced quinone, stigmatellin, or 5-(n-un decyl)-6-hydroxy-4,7-dioxobenzothiazole was present. Occupancy of the Q(o)- site by myxothiazole induced appearance of a minority population with a sub stantially differing conformation and presence of E-beta-methoxyacrylate-st ilbene significantly reduced the contribution of the major conformation obs erved in the other cases. Furthermore, when the oxidized iron-sulfur cluste r was reduced at cryogenic temperatures by the products of radiolysis, the orientation of its magnetic axes was found to differ significantly from tha t of the chemically reduced center. The "irradiation-induced" conformation converts to that of the chemically reduced center after thawing of the samp le. These results confirm the effects of Q(o)-site inhibitors on the equili brium conformation of the Rieske iron-sulfur protein and provide evidence f or a reversible redox-influenced interconversion between conformational sta tes. Moreover, the data obtained with the iron-sulfur protein demonstrate t hat the conformation of "EPR-inaccessible" reduction states of redox center s can be studied by inducing changes of redox state at cryogenic temperatur es. This technique appears applicable to a wide range of comparable electro n transfer systems performing redox-induced conformational changes.