Resonance energy transfer between tryptophan 57 in the epsilon subunit andpyrene maleimide labeled gamma subunit of the chloroplast ATP synthase

The intrinsic fluorescence of the catalytic portion of the chloroplast ATP synthase (CF1) is quenched when cysteine 322, the penultimate amino acid of the gamma subunit, is specifically labeled with pyrene maleimide (PM). The epsilon subunit of CF1 contains the only two residues of tryptophan, which dominate the intrinsic fluorescence of unlabeled CF1. CF1 deficient in the epsilon subunit (CF1-epsilon) was reconstituted with mutant epsilon subuni ts in which phenylalanine replaced tryptophan at position 15 (epsilon W15F) and position 57 (epsilon W15/57F). CF1(epsilon W15F) containing a single t ryptophan, epsilon W57, was labeled with PM at gamma C322. Resonance energy transfer (RET) from epsilon W57 to PM on gamma C322 occurred with an effic iency of energy transfer of 20%. RET was also observed from epsilon W57 to PM attached to the disulfide thiols of the gamma subunit (gamma C199,205) w ith an efficiency of approximately 45%. The R-o (the distance at which the efficiency of energy transfer is 50%) for the epsilon W57 and PM donor/acce ptor pair is 30 Angstrom, indicating that both gamma C322 and gamma C199,20 5 must be within 40 Angstrom of epsilon W57. These RET measurements show th at both gamma C322 and gamma C199,205 are located near the base of the alph a/beta hexamer. This places the C-terminus of CF1 gamma much closer to epsi lon than hypothesized based on homology to crystal structures of mitochondr ial Fl. These new RET measurements also allow the alignment of the predicte d epsilon subunit structure. The orientation is similar to that predicted f rom cross-linking and mutational studies fat the epsilon subunit of Escheri chia coli F1.