Resonance Energy Transfer between Tryptophan 57 in the ε Subunit and Pyrene Maleimide Labeled γ Subunit of the Chloroplast ATP Synthase

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Abstract
The intrinsic fluorescence of the catalytic portion of the chloroplast ATP synthase (CF1) is quenched when cysteine 322, the penultimate amino acid of the γ subunit, is specifically labeled with pyrene maleimide (PM). The ε subunit of CF1 contains the only two residues of tryptophan, which dominate the intrinsic fluorescence of unlabeled CF1. CF1 deficient in the ε subunit (CF1-ε) was reconstituted with mutant ε subunits in which phenylalanine replaced tryptophan at position 15 (εW15F) and position 57 (εW15/57F). CF1(εW15F) containing a single tryptophan, εW57, was labeled with PM at γC322. Resonance energy transfer (RET) from εW57 to PM on γC322 occurred with an efficiency of energy transfer of 20%. RET was also observed from εW57 to PM attached to the disulfide thiols of the γ subunit (γC199,205) with an efficiency of approximately 45%. The Ro (the distance at which the efficiency of energy transfer is 50%) for the εW57 and PM donor/acceptor pair is 30 Å, indicating that both γC322 and γC199,205 must be within 40 Å of εW57. These RET measurements show that both γC322 and γC199,205 are located near the base of the α/β hexamer. This places the C-terminus of CF1 γ much closer to ε than hypothesized based on homology to crystal structures of mitochondrial F1. These new RET measurements also allow the alignment of the predicted ε subunit structure. The orientation is similar to that predicted from cross-linking and mutational studies for the ε subunit of Escherichia coli F1.

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