Vol. i89, No.‘~, 1992 December 30, 1992 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 1477-1433 VALINOMYCIN MODIFIES PHOSPHORESCENCE QUENCHING IN CYTOCHROME c OXIDASE Peter Butkoa, Jia Heb and Peter NichollsC Department of Biological Sciences, Brock University, St. Catharines, Ont. L2S 3A1,Canada Received November 2, 1992 The spectrum of resting cytochrome c oxidase is modulated by valinomycin addition, which induces a red shift of the Soret band. The enzyme is known both to fluoresce and phosphoresce, effects which can be modulated by certain protein reagents and quenchers. Valinomycin had little effect upon fluorescence at 335 nm, whether excited at 295 nm or 280 nm (tryptophans or both tryptophans and tyrosines, respectively). Phosphorescence at 445 nm was slightly enhanced upon the binding of valinomycin to the enzyme, suggestinga small conformational change accompanying the spectral shift of the heme groups. The quenching by nitrite of the phosphorescence excited at 260 nm, but not that excited at 295 nm, was diminished by valinomycin. This suggests that the valinomycin-induced conformational change may involve (i) a change in the accessibility of tyrosines to the quencher and/or (ii) a change in distribution of distancesand/or orientations between populations of tyrosines and tryptophans. 0 1992 Academic Press, IX. Valinomycin has been considered a ‘friendly’ ionophore that does not interact with membrane proton pumps, particularly with cytochrome oxidase, and has thus been used for years in research on reconstituted membrane systems. First indications of direct interactions between the enzyme and the ionophore appeared in the work of Steverding and Kadenbach [l, 21.The effect was subsequentlyconfirmed by Prochaska and Wilson [3] and investigated in more detail by Nicholls and He [4], both in solubilized enzyme and proteoliposomal oxidase. In the present work, we applied fluorescence and phosphorescence techniques to obtain further insight into the influence of the valinomycin binding on the conformation of cytochrome oxidase. a Present address:Institute for Biological Sciences (M-54), National Research Council, Ottawa, Ont. KlA OR6,Canada. b Present address: Department of Biochemistry, University of Western Ontario, London, Ont. N6A 5C1, Canada. ’ To whom correspondence should be addressed. 0006-291 X/92 S4.00 1477 Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any .$x-m rrserwd.