INTERACTION OF PHOTOREACTIVE AND FLUORESCENT NUCLEOTIDES WITH CHLOROPLAST COUPLING FACTOR-1

The photoreactive nucleotide 3′-O-[3-[N-(4-azido-2-nitrophenyl)amino]propionyl]-ATP is a substrate of heat-activated chloroplast coupling factor 1 (CF1) and can be incorporated stoichiometrically (1:1) into the latent enzyme. Photolysis of the isolated [3H]nucleotide-CF, complex and analysis with sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that the subunit was covalently labeled. The analogous photoreactive ADP nucleotide binds to different sites which are specific for ADP. Photolysis of a mixture of the tritium-labeled photoreactive ADP and CF, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that both the α and β subunits are covalently labeled, in approximately equal amounts. In both cases, covalent labeling inactivates the enzyme, but a clear-cut correlation between the extent of labeling and loss of activity could not be established. The fluorescent nucleotide 1, N6-etheno-ATP (e-ATP) also can be stoichiometrically (1:1) incorporated into the latent enzyme, with a concomitant quenching in the fluorescence of about 30%. If a tyrosine residue of the β subunit in the fluorescent nucleotide-CF1 complex is modified with 7-chloro-4-nitrobenz-2-oxa-l, 3-diazole (NBD-C1), the fluorescence is first quenched and later enhanced. The tyrosine modification increases the rate of dissociation of ε-ATP from the CF1, causing the enhancement of fluorescence, a decrease in fluorescence polarization, and an apparent decrease in the resonance energy transfer from e-ATP to tyrosine as the reaction proceeds. Extrapolation of the energy-transfer efficiency to zero time suggests that the e-ATP is bound 35 Å from the NBD-tyrosine residue. © 1979, American Chemical Society. All rights reserved.