Chemical Conformation of the Essential Glutamate Site of the -Ring within Thermophilic FF-ATP Synthase Determined by Solid-State NMR Based on its Isolated -Ring Structure.

Proton translocation through the membrane-embedded F component of F-type ATP synthase (FF) is facilitated by the rotation of the F -subunit ring (-ring), carrying protons at essential acidic amino acid residues. Cryo-electron microscopy (Cryo-EM) structures of FF suggest a unique proton translocation mechanism. To elucidate it based on the chemical conformation of the essential acidic residues of the -ring in FF, we determined the structure of the isolated thermophilic F (tF) -ring, consisting of 10 subunits, in membranes by solid-state NMR. This structure contains a distinct proton-locking conformation, wherein Asn23 (N23) CO and Glu56 (E56) COH form a hydrogen bond in a closed form. We introduced stereo-array-isotope-labeled (SAIL) Glu and Asn into the tF-ring to clarify the chemical conformation of these residues in tFF-ATP synthase (tFF). Two well-separated C signals could be detected for N23 and E56 in a 505 kDa membrane protein complex, respectively, thereby suggesting the presence of two distinct chemical conformations. Based on the signal intensity and structure of the tF-ring and tFF, six pairs of N23 and E56 surrounded by membrane lipids take the closed form, whereas the other four in the - interface employ the deprotonated open form at a proportion of 87%. This indicates that the - interface is highly hydrophilic. The p values of the four E56 residues in the - interface were estimated from the N23 signal intensity in the open and closed forms and distribution of polar residues around each E56. The results favor a rotation of the -ring for ATP synthesis.