Chemical Conformation of the Essential Glutamate Site of the c-Ring within Thermophilic Bacillus FoF1-ATP Synthase Determined by Solid-State NMR Based on its Isolated c-Ring Structure

Proton translocation through the membrane-embedded Fo component of F-type ATP synthase (FoF1) is facilitated by the rotation of the Fo c-subunit ring (c-ring), carrying protons at essential acidic amino acid residues. Cryo-electron microscopy (Cryo-EM) structures of FoF1 suggest a unique proton translocation mechanism. To elucidate it based on the chemical conformation of the essential acidic residues of the c-ring in FoF1, we determined the structure of the isolated thermophilic Bacillus F-o(tF(o)) c-ring, consisting of 10 subunits, in membranes by solid-state NMR. This structure contains a distinct proton-locking conformation, wherein Asn23 (cN23) (CO)-O-gamma and Glu56 (cE56) (COH)-O-delta form a hydrogen bond in a closed form. We introduced stereo-array-isotope-labeled (SAIL) Glu and Asn into the tFoc-ring to clarify the chemical conformation of these residues in tF(o)F(1)-ATP synthase (tF(o)F(1)). Two well-separated C-13 signals could be detected for cN23 and cE56 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 tFoc-ring and tF(o)F(1), six pairs of cN23 and cE56 surrounded by membrane lipids take the closed form, whereas the other four in the a-c interface employ the deprotonated open form at a proportion of 87%. This indicates that the a-c interface is highly hydrophilic. The pK(a )values of the four cE56 residues in the a-c interface were estimated from the cN23 signal intensity in the open and closed forms and distribution of polar residues around each cE56. The results favor a rotation of the c-ring for ATP synthesis.

Automated summary

Proton translocation through the Fo component of F-type ATP synthase is facilitated by the rotation of the c-subunit ring. The study determined the chemical conformation of essential acidic residues in the c-ring and elucidated a unique proton translocation mechanism. These findings provide insights into ATP synthesis.