INTRINSIC FLUORESCENCE OF THE CHLOROPLAST H-ATPASE()

We have examined the intrinsic fluorescence properties of a highly pur ified chloroplast H+-ATPase (CF0F3) preparation [R. D. Kirch and P. Gr aber (1992) Acta Physiol. Scand. 746, 9-12). Unlike the catalytic CF1 portion alone, CF0F1 fluorescence was dominated by tryptophan fluoresc ence both at 277-nm excitation, favoring tyrosine excitation, and at 2 95-nm excitation, favoring tryptophan excitation. A broad tryptophan f luorescence peak was observed with a maximum at around 335 nm and a br oad shoulder around 350 nm. Denaturation of and the enzyme complex wit h guanidine-HCl resulted in a significant increase (similar to 40%) in tyrosine fluorescence. The fluorescence spectrum (lambda(ex) = 295 nm ) of the inhibitory epsilon subunit isolated from CF1 resembled that o f CF1, indicating the presence of two tryptophan species located in di fferent environments. Fluorescence quenching by potassium iodide indic ated a substantial increase in the solvent accessibility of one of the two tryptophans following isolation of epsilon from CF1. Thus, when e psilon binds to CF1, a tryptophan residue becomes partially buried, pr obably at an interface between epsilon and another (possibly gamma) CF 1 subunit. Removal of the epsilon subunit from CF1 leads to an increas e in tyrosine fluorescence of a magnitude similar to that obtained upo n denaturation of the CF0F1 complex. The results suggest that the reve rsible association of the epsilon subunit with CF0F1 or with isolated CF1 may be monitored by following changes in the intrinsic fluorescenc e of the enzyme complex. (C) 1995 Academic Press, Inc.