Site-directed spin-labeling of the catalytic sites yields insight into structural changes within the F0F1-ATP synthase of Escherichia coli

Electron spin resonance (ESR) spectroscopy using site-specific cysteine spin-labeling of the catalytic nucleotide binding, sites of F-1-ATPase was employed to investigate conformational changes within the nucleotide binding sites of the enzyme. Mutant Escherichia coli F-1 that had been modified at position beta-Y331C with a spin label showed almost normal catalytic activity and enabled us to study the effects of binding of different nucleotides and of the F-o subunit b on the conformation of the catalytic binding sites. The ESR spectra of the spin-labeled, nucleotide-depleted F-1 indicate asymmetry within the sites as is expected from the structural models of the enzyme. Nucleotide binding to the enzyme clearly affects the conformation of the sites; the most pronounced feature upon nucleotide binding is the formation of catalytic site(s) in a very open conformation. Using the same beta-331 spin-labeled F-1 and a truncated form of F-o subunit b, b(24-156), We found that binding of b(24-156) to spin-labeled F-1 significantly changes the conformation of the catalytic sites. Ln this paper we present data that for the first time directly show that a conformational binding change takes place upon binding of nucleotides to the nucleotide binding sites and that also show that binding of b(24-156) strongly affects the conformation of the catalytic sites, most likely by increasing the population of binding sites that are in the open conformation.