Interaction of 4-azido-2-nitrophenyl phosphate, an inorganic phosphate photoreactive analog, with chloroplast coupling factor 1

Abstract

4-Azido-2-nitrophenyl phosphate (ANPP), a photoreactive derivative of inorganic phosphate (Pj) [Lauquin, G., Pougeois, R., & Vignais, P. V. (1980) Biochemistry 19, 4620-4626], was used to study the P, binding site in purified spinach chloroplast coupling factor 1 (CFj). Upon photoirradiation with visible light, [32P]ANPP bound covalently to CF, and inactivated the enzyme. The labeling stoichiometry was measured in the presence of either EDTA, Mg2+, or Ca2+.

Demonstration of two binding sites for ADP on the isolated β‐subunit of the Rhodospirillum rubrum R1F0F1‐ATP synthase

“…Our observation, that the Mg-ADP-fi complex is unstable, dissociating very rapidly upon removal of MgClt after the binding step, might explain earlier reported data on the poor binding of nucleotides to the isolated E. co/i &subunit [5,9] and of Pi to CFi [19,20], if also in these cases an unstable Mg-ligand-protein complex is formed. indeed, in all these reports MgClz, even when present during the incubation of the protein with the Iigand, was removed when separating the bound and free ligands.…”

ATP Synthase and the Actions of Inhibitors Utilized To Study Its Roles in Human Health, Disease, and Other Scientific Areas

“…Phosphate added before photoirradiation protects the photoinactivation by ANPP. The stoichiometry for full photoinactivation of F 1 is approximately 1 mol of ANPP/mol of CF 1 (321).…”

Photolabeling of the phosphate binding site of chloroplast coupling factor 1 With [32P]azidonitrophenyl phosphate

“…Either P= and sulfite induce a high energy state similar to that resulting from light-induced generatio:,~ of a proton gradient [4] or they facilitate the conformational change induced by MgATP binding at another catalytic site [5]. ANPP was previously utilized in studies directed to the identification of the amino acid residues iavolved in the P= binding site of F,-ATPases from mitochondria [7,8], E. colt and PS3 [15] and the mitochondrial P= carrier [22], ANPP was also tbund to be a potential probe for CF~ [6]. In the present study, the target residues at the P~ binding site of CF~ have been investigated.…”

“…Although there are some data concerning P~ binding in connection with the photophosphorylation activity of membrane bound CF~ as well as ~he capacity of CF~ to synthesize bound ATP from bound ADP and e~ogenous P~ ( [2,3] and references therein), there was a need for a probe specific to the P~ binding site to confirm the tentative structural assignment of this site next to the tight ADP binding site of CF~ [4,5]. A previous study involving the photoactivatable P~ analogue 2-azido-4-nitrophenyl phosphate (ANPP) had led to the localization of this site on the,0 subu~fit [6]. The goal of the present work was to identify the amino acid residues of the ~ subunit which are labeled after photoirradiation of isolated chloroplast Ft ' chloroplast l~./l TPase Spinach CF~ wa~ purified as described by tlere, er et al, [gJand stored in 2 M ammonium sulfate, 10 mM Tris-HCl, I mM ED'I'A and 0,5 mM ATP, pH 7,2. at 4°C, The CFj sttsperl~ion in ammonium sulrate was eentrlfu~ed and the pellet was rinse.l with 250 mM sucrose, 50 ham Tris-aeetate, pH 7.5 (STA hurler) in the presence of 50% ammonium sulfate, and then resu~pended in STA buffer.…”