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

The kinetics of nucleotide binding to spinach chloroplast coupling factor CF 1 in a fully inhibited state were investigated by stopped‐flow experiments using the fluorescent trinitrophenyl analogue (NO 2 ) 3 Ph‐ADP. The CF 1 was in a state in which two of the three binding sites on the β subunits were irreversibly blocked with ADP, Mg 2+ and fluoroaluminate, while the three binding sites on the α 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. (NO 2 ) 3 Ph‐ADP binding to the remaining site on the third β subunit was characterised by a high dissociation rate of 15 s −1 , leading to a very low affinity (dissociation constant higher than 150 μM). Subsequent to isolation, CF 1 preparations contained two endogenously bound nucleotides. Pre‐loading with ATP yielded CF 1 with five tightly bound nucleotides and one free nucleotide‐binding site on a β subunit. Pre‐loading with ADP, however, resulted in a CF 1 preparation containing four tightly bound nucleotides and two free nucleotide binding sites. One of the two free binding sites was located on a β subunit, while the other was probably located on an α subunit.