Features of F-1-ATPase catalytic and noncatalytic sites revealed by fluorescence lifetimes and acrylamide quenching of specifically inserted tryptophan residues

Catalytic and noncatalytic nucleotide sites of the Fl sector of ATP synthas e were characterized by tryptophan fluorescence techniques. Seven Trp resid ues inserted in varied microenvironments in the catalytic sites, and one in the noncatalytic sites, were studied in mutant Fl enzymes which were other wise devoid of Trp. Parameters measured were fluorescence lifetimes and dyn amic and static quenching by acrylamide in the absence or presence of nucle otide. The results indicated that the solution structures of the mutant enz ymes were consistent with reported crystal structures. In enzyme with three empty noncatalytic sites, all sites were relatively inaccessible to acryla mide, indicating a dosed conformation. In contrast, when the three catalyti c sites were empty, they were relatively and equally accessible to acrylami de, indicating an open conformation. This was the case in the presence or a bsence of Mg2+. Residue beta-Trp-331 has been extensively used previously t o determine nucleotide binding parameters in F-1. Results here showed that in beta Y331W mutant F-1, each of the three beta-Trp-331 residues has an un usually long fluorescence lifetime, confirming that each contributes equall y to the overall fluorescence signal.