Intrinsic Fluorescence of the Chloroplast H+-ATPase

We have examined the intrinsic fluorescence properties of a highly purified chloroplast H+-ATPase (CF0F1) preparation [R. D. Kirch and P. Graber (1992) Acta Physiol. Scand. 746, 9-12). Unlike the catalytic CF1 portion alone, CF0F1 fluorescence was dominated by tryptophan fluorescence both at 277-nm excitation, favoring tyrosine excitation, and at 295-nm excitation, favoring tryptophan excitation. A broad tryptophan fluorescence peak was observed with a maximum at around 335 nm and a broad shoulder around 350 nm. Denaturation of and the enzyme complex with guanidine-HCl resulted in a significant increase (∼ 40%) in tyrosine fluorescence. The fluorescence spectrum (λex = 295 nm) of the inhibitory ϵ subunit isolated from CF1 resembled that of CF1, indicating the presence of two tryptophan species located in different environments. Fluorescence quenching by potassium iodide indicated a substantial increase in the solvent accessibility of one of the two tryptophans following isolation of ϵ from CF1. Thus, when ϵ binds to CF1, a tryptophan residue becomes partially buried, probably at an interface between ϵ and another (possibly γ) CF1 subunit. Removal of the ϵ subunit from CF1 leads to an increase in tyrosine fluorescence of a magnitude similar to that obtained upon denaturation of the CF0F1 complex. The results suggest that the reversible association of the ϵ subunit with CF0F1 or with isolated CF1 may be monitored by following changes in the intrinsic fluorescence of the enzyme complex.