NATURE CHEMICAL BIOLOGY | VOL 6 | DECEMBER 2010 | www.nature.com/naturechemicalbiology 891 ARTICLE PUBLISHED ONLINE: 24 OCTOBER 2010 | DOI: 10.1038/NCHEMBIO.457 F -type ATP synthases are large multisubunit complexes that use the energy stored in electrochemical gradients of H + (ref. 1) or Na + (ref. 2) for the synthesis of ATP, the universal chemi- cal energy source of living cells. The enzyme is composed of two structurally and functionally distinct parts; the catalytic F 1 complex (subunits α 3 β 3 γδε) and the membrane-embedded F o complex (for example, subunits ab 2 c 10–15 in bacteria), which conducts ions across the membrane. The mechanism of the enzyme entails the rotation of a subset of subunits against the others 3 ; these are collectively known as the rotor (γεc 10–15 ) and stator subcomplexes (α 3 β 3 δab 2 ). In ATP synthesis mode, the downhill movement of ions causes the rotation of the c-subunit assembly in F o , and therefore of subunit γ as well. This γ-subunit, referred to as the central stalk, protrudes into the α 3 β 3 assembly in F 1 (ref. 4); its rotation drives a cycle of conforma- tional changes within the catalytic β-subunits 5 , finally resulting in ATP synthesis and release. Conversely, F 1 can convert the chemical energy derived from ATP hydrolysis into a torque that drives the F o motor to act as an ion pump, in analogy with the related family of V 1 V o ATPases 6 . The c-subunit assembly in F o , known as the c-ring, is thus the key element that transduces the electrochemical energy into mechanical rotation and vice versa. It consists of a species-dependent number of c-subunits 7,8 and adopts a cylinder-like shape with a central, lipid- plugged pore. High-resolution crystal structures of the Na + -coupled c 11 ring from Ilyobacter tartaricus 9,10 and the K 10 ring from Enterococcus hirae 11 , and of the H + -coupled c 15 ring from Spirulina platensis 12 and the c 13 ring from Bacillus pseudofirmus OF4 (ref. 13) have been obtained. In all structures the ion-binding sites are located near the outer surface of the ring, facing the hydrophobic acyl-chain layer of the membrane. The ions bind to grooves formed between adjacent c-subunits and establish a precise network of interactions; a strictly conserved carbox- ylate residue is the key ligand for both Na + and H + . In these structures, the binding sites are observed in the ion-locked state 9,12,13 . The microscopic mechanism by which ion translocation across the F o complex is coupled to the rotation of the c-ring is not known. Nonetheless, it is widely accepted that two independent, discon- nected access pathways probably exist at the interface of the c-ring and the a-subunit or within the latter, which allow H + or Na + ions to bind to or be released from the c-subunit rotor. The bound ions would be shuttled from one access channel to the other as the c-ring rotates around its central axis, effectively completing the ion pathway across the membrane 14,15 . The underlying principle of this mechanism is that the occupancy of the binding sites in the c-ring is dependent on the chemical environment they face at a given time. As protons (or Na + ) are shuttled within the membrane core by the rotating ring, they remain tightly bound to the c-ring. By contrast, the environment of the a-subunit enables these binding sites to load and release ions from and to the access channels, through a mecha- nism that has not been characterized in detail. This is partly because of the lack of a high-resolution structure of the a-subunit and its interface with the c-ring. Nevertheless, biochemical data have established two important facts: first, that the a-subunit harbors a strictly conserved arginine, whose guanidine group is believed to have a central role as an electrostatic separator between these access pathways 14,15 , and second, that the a-c interface is locally hydrated from the cytoplasmic side to the membrane middle, as evidenced by solvent-accessibility measurements specific to this region 16 . Lately, electron microscopy data on a related V-type ATPase 17 has indi- cated that the contact interface of subunit a and the c-ring (or their equivalent) is indeed rather minimal and that it appears to include not only water but lipids as well. In this work we use experimental and theoretical methods to show how these essential features give rise to a mechanism of envi- ronmental control of the ion occupancy of the c-subunit and thus to the ion-coupled rotary mechanism within the F o domain. As sup- porting evidence, we provide three atomic-resolution structures of the H + -coupled c 15 ring rotor from S. platensis, together with a sys- tematic, quantitative DCCD–modification assay and detailed free- energy molecular-dynamics calculations. Our results support the view that the membrane and a-subunit environments influence the 1 Department of Structural Biology, Max Planck Institute of Biophysics, Frankfurt am Main, Germany. 2 Theoretical Molecular Biophysics Group, Max Planck Institute of Biophysics, Frankfurt am Main, Germany. 3 Department of Molecular Membrane Biology, Max Planck Institute of Biophysics, Frankfurt am Main, Germany. 4 Cluster of Excellence Macromolecular Complexes, Frankfurt am Main, Germany. 5 These authors contributed equally to this work. *e-mail: thomas.meier@biophys.mpg.de or jose.faraldo@biophys.mpg.de Microscopic rotary mechanism of ion translocation in the F o complex of ATP synthases Denys Pogoryelov 1 , Alexander Krah 2 , Julian D Langer 3 , Özkan Yildiz 1 , José D Faraldo-Gómez 2,4,5 * & Thomas Meier 1,4,5 * The microscopic mechanism of coupled c-ring rotation and ion translocation in F 1 F o -ATP synthases is unknown. Here we present conclusive evidence supporting the notion that the ability of c-rings to rotate within the F o complex derives from the interplay between the ion-binding sites and their nonhomogenous microenvironment. This evidence rests on three atomic structures of the c 15 rotor from crystals grown at low pH, soaked at high pH and, after N,N′-dicyclohexylcarbodiimide (DCCD) modification, resolved at 1.8, 3.0 and 2.2 Å, respectively. Alongside a quantitative DCCD-labeling assay and free-energy molecular dynamics calculations, these data demonstrate how the thermodynamic stability of the so-called proton-locked state is maximized by the lipid membrane. By contrast, a hydrophilic environment at the a-subunit–c-ring interface appears to unlock the binding-site conformation and promotes proton exchange with the surrounding solution. Rotation thus occurs as c-subunits stochastically alternate between these environments, directionally biased by the electrochemical transmembrane gradient. © 2010 Nature America, Inc. All rights reserved.