(proton pumps/proton translocation/chemiosmosis/acid membranes/acid-anions)

Evidence has been gathering from several laboratories that protons in proton-pumping membranes move along or within the bilayer rather than exchange with the bulk phase. These experiments are typically conducted on the natural membrane in vivo or in vitro or on fragments of natural membrane. Anionic lipids are present in all proton-pumping membranes. Model studies on the protonation state of the fatty acids of liposomes containing entrapped water show that the bilayers always contain mixtures of protonated and deprotonated carboxylates. Protonated fatty acids form stable acid-anion pairs with deprotonatedfatty acids through unusually strong hydrogen bonds. Such acid-anion dimers have a single negative charge, which is shared by the four negative oxygens of both headgroups. The two pK values of the resulting dimer will be significantly different from the pK of the monomeric species, so that the dimer will be stable over a wide pH range. It is proposed that anionic lipid headgroups in biological membranes share protons as acid-anion dimers and that anionic lipids thus trap and conduct protons along the headgroup domain of bilayers that contain such anionic lipids-. Protons pumped from the other side ofthe membrane may enter and move within the headgroup sheet because the protonation rate of negatively charged proton acceptors is 5 orders of magnitude faster than that of water. Protons trapped in the acidic headgroup sheet need not leave this region in order to be utilized by a responsive proton-translocating pore (a transport protein using the proton gradient). Experiments suggest the proton concentration in the headgroup domain may vary widely and the anionic lipid headgroup sheet may therefore function as a proton buffer. -Due to the Gouy-Chapman-Stern layer at polyanionic surfaces, anionic lipids will also sequester protons from the bulk solution at low and moderate ionic strengths. At high ionic strength metal cations may replace protons sequestered near the headgroups, but these cations cannot substitute for protons in the "proton-conducting pathway," which is based on hydrogen bonding. Recent experiments in several laboratories have suggested that protons that are translocated by proton-pumping membranes, such as those of mitochondria and halobacteria, need not enter the external bulk water phase in order to be utilized by protontransporting proteins ofthe membrane, such as ATP synthetase. Notable among these experiments are -those of Michel and Oesterhelt (1), who made careful measurements of proton-dependent ATP synthesis and pH changes in and near illuminated, Halobacterium halobium cells. They found, that the pH is not lowered in the bulk aqueous phase as the protons pumped.by the purple patches of bacteriorhodopsin are utilized by ATP synthetase, which is not present in the purple patches but is found at some distance in red patches of the same plasma membrane. They concluded that.the protons do not pass through the bulk aqueous phase but move very close to the membrane surface. This system is unique because the organism is cultured in 6 M NaCl, which implies that the Gouy-Chapman-Stern layer contains exclusively Na+ and is not rich in H30+. Other reports (2-18) have described proton movements in restricted domains or in isolated open membrane sheets not capable of sustaining a transmembrane pH gradient (which nevertheless can be energized, ostensibly by generation of an electrochemical H+ gradient for ATP synthesis) or have demonstrated that the pH may not account for the activities attributed to membrane "energization." Taken together, the observations suggest that there may be specific proton-conducting pathways in or on such energy-transducing membranes that allow them to carry out energy transduction without necessarily involving a pH gradient across the membrane. Considerations developed in this paper suggest that the anionic headgroups of the lipids of energy-transducing membranes have the capacity to bind and conduct protons along the membrane surface. It is proposed that anionic groups, such as phosphodiester anions, form acid-anion dimers, two anions sharing a single proton via a hydrogen bond. Evidence is reviewed suggesting that such putative acid-anion dimers may be protonated and deprotonated at a high rate, permitting them to constitute a proton-conducting sheet or continuum, rapidly moving protons through the headgroups (along the surface) from H+-pumping (e.g., electron transport chain protein) and H+utilizing systems (ATP synthetases) anywhere in the membrane. Protons trapped in such a proton-conducting continuum can, eventually, equilibrate with the bulk aqueous phase. This may account for the capacity of an imposed pH gradient to be used experimentally to generate ATP in proton-pumping membrane vesicles. These suggestions came from experimental studies designed to explain the structure and dynamics ofthe flagellar membrane of the phytoflagellate Ochromonas danica. We had focused on the protonation state of the primary and secondary sulfates of the docosanediol 1, 14-disulfate and the series ofanalogues containing one to six chloro groups on an otherwise saturated chain (19-28). This unusual natural membrane is devoid of phospholipids. This type of membrane occurs in many freshwater algae (29, 30) and may represent a-diversion in the evolution of biological membranes (31). Natural membranes made of anionic detergents are surprising, more so when the detergent structure suggests that their anionic groups may be buried in the hydrophobic domain of the bilayer. Elemental analyses of such membrane preparations (unpublished data) excluded all potential counterions except for the proton. We therefore instituted studies on.model bilayers containing alkyl sulfates or fatty acids in an attempt to understand how protons may stabilize such membranes and participate in their function. Fatty Acid Liposomes.. Unsaturated fatty acid liposomes that entrap aqueous compartments were described by Gebicki and Hicks 10years ago (32). Liposomes may also be made of satu160 The publication costs ofthis article were defrayed in part by page charge' payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact. Proc. Natl. Acad. Sci. USA 80 (1983) 161