Active-Site Structure of the Thermophilic Foc-Subunit Ring in Membranes Elucidated by Solid-State NMR

F_oF₁-ATP synthase uses the electrochemical potential across membranes or ATP hydrolysis to rotate the F_oc-subunit ring. To elucidate the underlying mechanism, we carried out a structural analysis focused on the active site of the thermophilic c-subunit (TF_oc) ring in membranes with a solid-state NMR method developed for this purpose. We used stereo-array isotope labeling (SAIL) with a cell-free system to highlight the target. TF_oc oligomers were purified using a virtual ring His tag. The membrane-reconstituted TF_oc oligomer was confirmed to be a ring indistinguishable from that expressed in E. coli on the basis of the H⁺-translocation activity and high-speed atomic force microscopic images. For the analysis of the active site, 2D ¹³C-¹³C correlation spectra of TF_oc rings labeled with SAIL-Glu and -Asn were recorded. Complete signal assignment could be performed with the aid of the C^α_i+1-C^α_i correlation spectrum of specifically ¹³C,¹⁵N-labeled TF_oc rings. The C^δ chemical shift of Glu-56, which is essential for H⁺ translocation, and related crosspeaks revealed that its carboxyl group is protonated in the membrane, forming the H⁺-locked conformation with Asn-23. The chemical shift of Asp-61 C^γ of the E. coli c ring indicated an involvement of a water molecule in the H⁺ locking, in contrast to the involvement of Asn-23 in the TF_oc ring, suggesting two different means of proton storage in the c rings.