PHOTOLABELING OF MITOCHONDRIAL F(1)-H-AZIDO[H-3]ADP AND 8-AZIDO[H-3]ADP ENTRAPPED AS FLUOROMETAL COMPLEXES INTO THE CATALYTIC SITES OF THE ENZYME( ATPASE BY 2)

In the presence of ADP and fluorometals, the ATPase activity of the ca talytic sector, F1, of beef heart mitochondrial ATPase is strongly inh ibited; this inhibition is dependent on the entrapment of ADP-fluoroal uminate complexes into the nucleotide binding sites of F1 [Lunardi, J. , Dupuis, A., Garin, J., Issartel, J. P., Michel, L., Chabre, M., & Vi gnais, P. V. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 8958-8962]. We d escribe here the effect of fluoroaluminate on the binding of 2-azido[H -3]ADP and 8-azido[H-3]ADP to beef heart mitochondrial F1 in the absen ce and presence of light. When the incubation medium was supplemented with NaF and AlCl3, and maintained in the dark, both 2-azido[H-3]ADP a nd 8-azido[H-3]ADP were able to elicit inhibition of F1-ATPase activit y, exactly like ADP did. Upon photoirradiation, 2-azido[H-3]ADP and 8- azido[H-3]ADP bound covalently to F1. Labeling was restricted to the b eta subunit of F1, and the same tyrosine residue, beta-Tyr-345, was la beled by either of the photoprobes. This is in contrast with the previ ous findings that in the absence of fluoroaluminate both the alpha and beta subunits of F1 were photolabeled by 8-azido[H-3]ADP, and that tw o different regions of the beta subunit were labeled, centered on beta -Tyr-345 in the case of 2-azido[H-3]ADP [Garin, J., Boulay, F., Issart el, J. P., Lunardi, J., & Vignais, P. V. (1986) Biochemistry 25, 4431- 4437] and beta-Tyr-311 in that of 8-azido[H-3]ADP [Hollemans, M., Runs wick, M., Fearnley, 1. H., & Walker, J. E. (1983) J. Biol. Chem. 258, 9307-9313]. It is postulated that fluoroaluminate nucleotide complexes promote a rearrangement of the architecture of the catalytic site of F1 that enables the two opposite sides of the adenine ring of the boun d nucleotide to interact with the same peptide segment of the beta sub unit.