Protonmotive force (the transmembrane difference in electrochemical potential of protons, ) drives ATP synthesis in bacteria, mitochondria, and chloroplasts. It has remained unsettled whether the entropic (chemical) component of relates to the difference in the proton activity between two bulk water phases (deltapH(B)) or between two membrane surfaces (deltapH(S)). To scrutinize whether deltapH(S) can deviate from deltapH(B), we modeled the behavior of protons at the membrane/water interface. We made use of the surprisingly low dielectric permittivity of interfacial water as determined by O. Teschke, G. Ceotto, and E. F. de Souza (O. Teschke, G. Ceotto, and E. F. de Sousa, 2001, PHYS: Rev. E. 64:011605). Electrostatic calculations revealed a potential barrier in the water phase some 0.5-1 nm away from the membrane surface. The barrier was higher for monovalent anions moving toward the surface (0.2-0.3 eV) than for monovalent cations (0.1-0.15 eV). By solving the Smoluchowski equation for protons spreading away from proton "pumps" at the surface, we found that the barrier could cause an elevation of the proton concentration at the interface. Taking typical values for the density of proton pumps and for their turnover rate, we calculated that a potential barrier of 0.12 eV yielded a steady-state pH(S) of approximately 6.0; the value of pH(S) was independent of pH in the bulk water phase under neutral and alkaline conditions. These results provide a rationale to solve the long-lasting problem of the seemingly insufficient protonmotive force in mesophilic and alkaliphilic bacteria.

The membrane portion of F(0)F(1)-ATP synthase, F(0), translocates protons by a rotary mechanism. Proton conduction by F(0) was studied in chromatophores of the photosynthetic bacterium Rhodobacter capsulatus. The discharge of a light-induced voltage jump was monitored by electrochromic absorption transients to yield the unitary conductance of F(0). The current-voltage relationship of F(0) was linear from 7 to 70 mV. The current was extremely proton-specific (>10(7)) and varied only slightly ( approximately threefold) from pH 6 to 10. The maximum conductance was approximately 10 fS at pH 8, equivalent to 6240 H(+) s(-1) at 100-mV driving force, which is an order-of-magnitude greater than of coupled F(0)F(1). There was no voltage-gating of F(0) even at low voltage, and proton translocation could be driven by deltapH alone, without voltage. The reported voltage gating in F(0)F(1) is thus attributable to the interaction of F(0) with F(1) but not to F(0) proper. We simulated proton conduction by a minimal rotary model including the rotating c-ring and two relay groups mediating proton exchange between the ring and the respective membrane surface. The data fit attributed pK values of approximately 6 and approximately 10 to these relays, and placed them close to the membrane/electrolyte interface.

ATP synthase couples transmembrane proton transport, driven by the proton motive force (pmf), to the synthesis of ATP from ADP and inorganic phosphate (P i ). In certain bacteria, the reaction is reversed and the enzyme generates pmf, working as a proton-pumping ATPase. The ATPase activity of bacterial enzymes is prone to inhibition by both ADP and the C-terminal domain of subunit ⑀. We studied the effects of ADP, P i , pmf, and the C-terminal domain of subunit ⑀ on the ATPase activity of thermophilic Bacillus PS3 and Escherichia coli ATP synthases. We found that pmf relieved ADP inhibition during steady-state ATP hydrolysis, but only in the presence of P i . The C-terminal domain of subunit ⑀ in the Bacillus PS3 enzyme enhanced ADP inhibition by counteracting the effects of pmf. It appears that these features allow the enzyme to promptly respond to changes in the ATP:ADP ratio and in pmf levels in order to avoid potentially wasteful ATP hydrolysis in vivo.ATP synthase (F O F 1 ) is a ubiquitous enzyme present in the plasma membrane of bacteria, the thylakoid membrane of chloroplasts, and the inner mitochondrial membrane. The enzyme catalyzes ATP synthesis coupled to transmembrane proton translocation 2 driven by the proton motive force (pmf).3 At low pmf the activity is reversed and the enzyme functions as a proton-pumping ATPase. ATP synthase consists of two distinct regions: the hydrophobic F O , which is embedded in the membrane, and the hydrophilic F 1 that protrudes ϳ100 Å from the membrane bilayer. F 1 contains three catalytic and three non-catalytic nucleotide-binding sites and is responsible for both ATP synthesis and hydrolysis reactions. F O performs transmembrane proton transport. The simplest subunit composition is found in bacteria (e.g. Escherichia coli or thermophilic Bacillus PS3; see Refs. 1 and 2 for reviews), where F 1 is a complex of five types of subunits at a stoichiometry of ␣ 3 ␤ 3 ␥ 1 ␦ 1 ⑀ 1 , and F O is a complex of three types of subunits at a stoichiometry of a 1 b 2 c 10 .It is now widely accepted that the enzyme operates according to the "binding change mechanism" (see Ref. 3 and the references therein). Briefly, proton translocation is coupled to the rotation of the c-ring oligomer (4) together with the ␥⑀ complex relative to the rest of the enzyme (see Refs. 5-7 and references therein for details). Rotation of the ␥ subunit inside the ␣ 3 ␤ 3 hexamer causes sequential conformational changes to the catalytic sites. These sequential conformational changes result in substrate binding, the chemical step, and product release (8 -10). This "rotary binding change mechanism" is usually considered reversible, and numerous demonstrations of ATPdriven proton pumping support this assumption. However, several observations indicate that this is not the case. Many factors (e.g. products/substrates of catalysis, inhibitors, inorganic anions, etc.) affect ATP synthesis and the hydrolysis activities of the enzyme (see Ref. 11 for a detailed discussion). One of the most well known anisotropic reg...