Solution Structure of the Transmembrane H<sup>+</sup>-Transporting Subunit c of the F<sub>1</sub>F<sub>o</sub> ATP Synthase<sup>†</sup>

Subunit c is the H<sup>+</sup>-translocating component of the F<sub>1</sub>F<sub>o</sub> ATP synthase complex. H<sup>+</sup> transport is coupled to conformational changes that ultimately lead to ATP synthesis by the enzyme. The properties of the monomeric subunit in a single-phase solution of chloroform−methanol−water (4:4:1) have been shown to mimic those of the protein in the native complex. Triple resonance NMR experiments were used to determine the complete structure of monomeric subunit c in this solvent mixture. The structure of the protein was defined by >2000 interproton distances, 64 <sup>3</sup><i>J</i><sub>N</sub><sub>α</sub>, and 43 hydrogen-bonding NMR-derived restraints. The root mean squared deviation for the backbone atoms of the two transmembrane helices was 0.63 Å. The protein folds as a hairpin of two antiparallel helical segments, connected by a short structured loop. The conserved Arg41-Gln42-Pro43 form the top of this loop. The essential H<sup>+</sup>-transporting Asp61 residue is located at a slight break in the middle of the C-terminal helix, just prior to Pro64. The C-terminal helix changes direction by 30 ± 5° at the conserved Pro64. In its protonated form, the Asp61 lies in a cavity created by the absence of side chains at Gly23 and Gly27 in the N-terminal helix. The shape and charge distribution of the molecular surface of the monomeric protein suggest a packing arrangement for the oligomeric protein in the F<sub>o</sub> complex, with the front face of one monomer packing favorably against the back face of a second monomer. The packing suggests that the proton (cation) binding site lies between packed pairs of adjacent subunit c.