Characterization of a phosphate binding domain on the alpha-subunit of chloroplast ATP synthase using the photoaffinity phosphate analogue 4-azido-2-nitrophenyl phosphate

The photoaffinity phosphate analogue 4-azido-2 nitrophenyl phosphate (ANPP) was shown previously (Pougeois, R., Lauquin, G. J.-M., and Vignais, P. V, (1983) Biochemistry 22, 1241-1245) to bind covalently and specifically to a single catalytic site on one of the three beta-subunits of the isolated ch loroplast coupling factor I (CF1). Modification by ANPP strongly inhibited ATP hydrolysis activity. In this study, we examined labeling of membrane-bo und CF1 by ANPP by exposing thylakoid membranes to increasing concentration s of the reagent, ANPP exhibited saturable binding to two sites on CF1, one on the beta -subunit and one on the alpha-subunit. Labeling by ANPP result ed in the complete inhibition of both ATP synthesis and ATP hydrolysis by t he membrane-bound enzyme. Labeling of both sites by ANPP was reduced by mor e than 80% in the presence of P-i (greater than or equal to 10 mM) and ATP (greater than or equal to0.5 mM). ADP was less effective in competing with ANPP for binding, giving a maximum of similar to 35% inhibition at concentr ations greater than or equal to2 mM. ANPP-labeled tryptic peptides of the c r-subunit were isolated and sequenced. The majority of the probe was contai ned in three peptides corresponding to residues Gln(173) to Arg(216), Gly(2 17) to Arg(253) and His(256) to Arg(272) Of the alpha -subunit. In the mito chondrial F-1 (Abrahams, J. P., Leslie, A. G. W,, Lutter, R,, and Walker, J . E. (1994) Nature 370, 621-628), all three analogous peptides are located within the nucleotide binding pocket and within close proximity to the gamm a -phosphate binding site. The data indicate, however, that the azidophenyl group of bound ANPP is oriented at approximately 180 degrees in the opposi te direction to the adenine binding site with reference to the phosphate bi nding site on the alpha-subunit. The study has confirmed that ANPP is a bon a fide phosphate analogue and suggests that it specifically targets the gam ma -phosphate binding site within the nucleotide binding pockets on the alp ha- and beta -subunits of CF1. The study also indicates that in the resting state of the chloroplast F-1-F-0 complex both the alpha- and beta -subunit s are structurally asymmetric.