Localization of the nucleotide binding sites on subunits of CF₁ and TF₁ using the photoreactive nucleotide analog 3-O-(4-benzoyl) benzoyl ATP

Abstract

The F₁F_O proton translocating ATP synthetases¹ from mitochondrial, chloroplast thylakoids and bacterial plasma membranes are the principal catalyst in the transduction of energy derived from electron transport (1–3). These enzyme complexes are generally composed of an integral membrane sector, F_O, which mediates H⁺ translocation, and a peripheral protein sector, F₁ which catalyzes the terminal step of ATP synthesis from ADP and P_i. F₁ is composed of five different polypeptide chains designated α-ε in order of decreasing M_r. The molecular weight of F₁ is about 380, 000 and the subunit stoichiometry is α₃β₃γδε. The binding sites for ADP and ATP are located on the α and β subunits and the catalytic sites are thought to be on the β subunits. The F₁-ATPases from mitochondria (MFj) and E. coli (EF₁) were shown to contain six nucletide binding sites (1, 2). The F₁-ATPase from the thermophilic bacterium, PS3 (TF₁), contains at least four nucleotide binding sites (4) and CF₁, from chloroplast thylakoid membranes, contains three nucleotide binding sites (5, 6). TF₁ has three noncatalytic nucleotide binding sites putatively in the α subunits and at least one catalytic site on the β subunit (4). Soluble CF₁ has two sites where ADP can bind, and in the presence of Mg²⁺ and ATP, another single site for ATP was described (6). Tight binding of ADP and ATP to the membrane bound CF₁ was also described (7–10). Although the role of these nucleotide binding sites has been studied extensively, it is difficult to distinguish between nucleotides bound at catalytic and noncatalytic sites. Several lines of evidence amply documented by Boyer and coworkers (7) support the participation of tightly bound nucleotides as catalytic intermediated in ATP synthesis and hydrolysis.