Metals as a probe of 'Rieske' ISP domain movements

Abstract

We have recently shown (Rao BK, S., Tyryshkin, A.M., Roberts, A.G., Bowman, M.K. and Kramer, D.M. (2000) Biochemistry 39, 3285-3296) that copper (Cu1+ and Cu2+) is a competitive inhibitor, with respect to substrate plastoquinol (PQH2), of the cytochrome (cyt) b6f complex, but that it binds at a histidine that is far from the quinol oxidase (Qo) site, most likely on the cyt f protein. We suggested that Cu binding interferes with PQH2 binding at the Qo site via long-range interactions, probably by altering the conformation of the soluble 'head' domain of the Rieske iron-sulfur protein (ISP), which is known to contribute thermodynamically to binding at the Qo site. In this work we demonstrate, through oriented EPR studies, that Cu2+/1+ and Zn2+ shift the conformation of the ISP head domain be in the same direction, but to a smaller extent, as stigmatellin and DBMIB. Thus, inhibitory metals place the ISP in intermediate positions, as observed in some X-ray structures and . This confirms our hypothesis that Cu and Zn are specific inhibitors of ISP conformational changes, strongly supporting models in which such changes are intrinsic to the Q-cycle. Also, as a part of this work, we present a structural model of the cyt b6f complex based on the published cyt bc1 structures and available cyt b6f data, and assign the g-transition of the 2Fe2S cluster to the structural axes. This work was supported by an award by the Frasch Foundation and by the US Department of Energy Grant DE-FG03-98ER20299.