Structural mapping of catalytic site with respect to alpha−subunit and noncatalytic site in yeast mitochondrial F1−ATPase using fluorescence resonance energy transfer

The intrinsic tryptophan fluorescence of Schizosaccharomyces pombe mitochondrial F1 is a very sensitive probe to differentiate nucleotide binding to catalytic and noncatalytic sites (Divita, G., Di Pietro, A., Roux, B., and Gautheron, D. C. (1992) Biochemistry 31, 5791−5798), the catalytic site saturation producing quenching of Trp−257 fluorescence (Divita, G., Jault, J.−M., Gautheron, D. C., and Di Pietro, A. (1993) Biochemistry 32, 1017−1024). The present results indicate that two types of fluorescent nucleotide analogues, bearing either 2'(3')N− methylanthraniloyl (mant) or 2',3'−O−(2,4,6−trinitrophenyl) (TNP) group, exhibit high−affinity binding and behave similarly to the corresponding unmodified nucleotides. Selective binding of mant GDP to the catalytic site produces a marked quenching of intrinsic fluorescence which is due to resonance energy transfer between Trp−257 and the mant group. The high efficiency of the transfer allows the determination of a short distance, 10.5 A, indicating the close proximity of catalytic site and alpha−subunit Trp−257. Selective saturation of the noncatalytic site by TNP−ADP produces a marked quenching of the extrinsic fluorescence of mant GDP bound to the catalytic site, which is correlated to an important resonance energy transfer between the two fluorescent groups. A rather short distance of 17.5 A is calculated, indicating vicinity of catalytic and noncatalytic site