FoF1-ATP synthase uses the electrochemical potential across membranes or ATP hydrolysis to rotate the Foc-subunit ring. To elucidate the underlying mechanism, we carried out a structural analysis focused on the active site of the thermophilic c-subunit (TFoc) 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. TFoc oligomers were purified using a virtual ring His tag. The membrane-reconstituted TFoc 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 (13)C-(13)C correlation spectra of TFoc 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 (13)C,(15)N-labeled TFoc 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 TFoc ring, suggesting two different means of proton storage in the c rings.

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helix, or adjacent alpha helices, depend on enzyme transport activity, with the adjoining extracellular loop favoring binding of positively-charged ions. One interpretation of these data is that conformational changes involving the first transmembrane alpha helix affect the disposition of H 2 O molecules in the membrane domain of the enzyme. These data, together with crystallographic data for other P-type ATPases, suggest that this region in the enzyme's transmembrane domain contains an extracellularly-facing cavity. We speculate that this cavity could contribute to a release pathway for H þ during the ion transport cycle.

Cytochrome c oxidase (CcO) is the terminal oxidase in the mitochondrial respiratory chain. It catalyzes the reduction reaction of the molecular oxygen, which is coupled with the proton-pumping. Azide is a potent inhibitor of CcO, and considered to bind to heme a 3 in the oxidized state. Thus far, there is no resonance Raman spectrum of azide-bound CcO. Here we report spectroscopic characterization of the interaction of azide with fully oxidized CcO. Addition of azide to CcO shifts the Soret peak and changes the intensity of the band, showing two azide-binding sites with K d values of 59 μM and 44 mM at pH 7.4. Resonance Raman analysis using 441.6 nm as the exciting laser shows very small spectral changes. The results suggest the absence of direct azide binding to heme a 3 .