Rotary catalysis mechanism of H^+-ATP synthase investigated by novel NMR methodology

点击翻译按钮获取中文摘要

F_OF_1-ATP synthase is a multisubunit enzyme that catalyzes ATP synthesis using the electrochemical potential of proton gradient. F_1-ATPase has been shown to be a step-wise molecular motor. The rotation mechanism has been explained by the interaction of the γ axis with the open and closed forms of the β subunit. Although NMR should be a powerful method to elucidate its mechanism, the molecular size (473 amino acid residues, 52 kDa) was the major obstacle to analyze. We have applied the segmental labeling based on intein ligation to the β subunit, and succeeded in assigning 89% NH (402/451),89% C_α (417/473),83% C_β(357/431),90% CO (425/473) signals of the β subunit monomer. The secondary structures predicted by the chemical shifts of the main chain atoms and the relative orientations determined by residual dipolar couplings indicated that the subunit β monomer takes the open form in the absence of nucleotide. Furthermore, the chemical shift perturbation and the residual-dipolar-coupling changes induced by nucleotide binding have shown the conformational change from the open to closed forms takes place on nucleotide binding. The intrinsic conformational change of the β subunit monomer induced by nucleotide binding must be one of essential driving forces of the rotation of F_1-ATPase. In F_OF_1-ATP synthase, an oligomer ring of F_Oc subunits acts as a rotary proton channel of F_O-proton motor. We have determined the NMR structure of F_Oc from thermophilic Bacillus PS3, TF_Oc in an organic solvent. Our results showed that, independent of pH, the carboxyl group of the essential Glu56 of TF_Oc protrudes to the outside of the hairpin, a third orientation differed from either of two orientations in EF_Oc. A solid-state NMR analysis of solid subunit c was also carried out.