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 mit ochondrial F1 is a very sensitive probe to differentiate nucleotide bi nding 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 ty pes of fluorescent nucleotide analogues, bearing either 2'(3')N-methyl anthraniloyl (mant) or 2',3'-O-(2,4,6-trinitrophenyl) (TNP) group, exh ibit high-affinity binding and behave similarly to the corresponding u nmodified nucleotides. Selective binding of mant GDP to the catalytic site produces a marked quenching of intrinsic fluorescence which is du e to resonance energy transfer between Trp-257 and the mant group. The high efficiency of the transfer allows the determination of a short d istance, 10.5 angstrom, indicating the close proximity of catalytic si te and alpha-subunit Trp-257. Selective saturation of the noncatalytic site by TNP-ADP produces a marked quenching of the extrinsic fluoresc ence of mant GDP bound to the catalytic site, which is correlated to a n important resonance energy transfer between the two fluorescent grou ps. A rather short distance of 17.5 angstrom is calculated, indicating vicinity of catalytic and noncatalytic sites.