Kinetic properties of a single nucleotide binding site on chloroplast coupling factor 1 (CF1)

The kinetics of nucleotide binding to spinach chloroplast coupling factor C F1 in a fully inhibited state were investigated by stopped-flow experiments using the fluorescent trinitrophenyl analogue (NO2)(3)Ph-ADP. The CF1 was in a state in which two of the three binding sites on the beta subunits wer e irreversibly blocked with ADP, Mg2+ and fluoroaluminate, while the three binding sites on the alpha subunits were occupied by nucleotides [Garin, J. , Vincon, M., Gagnon, J. & Vignais, P. V. (1994) Biochemistry 33, 3772-3777 )]. Thus, it was possible to characterise a single nucleotide-binding site without superimposed nucleotide exchange or binding to an additional site. (NO2)(3)Ph-ADP binding to the remaining site on the third beta subunit was characterised by a high dissociation rate of 15 s(-1), leading to a very lo w affinity (dissociation constant higher than 150 mu M). Subsequent to isol ation, CF1 preparations contained two endogenously bound nucleotides. Pre-l oading with ATP yielded CF1 with five tightly bound nucleotides and one fre e nucleotide-binding site on a beta subunit. Pre-loading with ADP, however, resulted in a CF1 preparation containing four tightly bound nucleotides an d two free nucleotide binding sites. One of the two free binding sites was located on a beta subunit, while the other was probably located on an alpha subunit.