Mechanism of Ion Transport across Membranes

The high resolution structures now available for mitochondrial ATPase (1Abrahams J.P. Leslie A.G.W. Lutter R. Walker J.E. Nature. 1994; 370: 621-628Crossref PubMed Scopus (2719) Google Scholar) and mammalian and bacterial cytochromec oxidases (2Tsukihara T. Aoyama H. Yamashita E. Tomizaki T. Yamaguchi H. Shinzawa-Itoh K. Nakashima R. Yaono R. Yoshikawa S. Science. 1995; 269: 1069-1074Crossref PubMed Scopus (1283) Google Scholar, 3Iwata S. Ostermeier C. Ludwig B. Michel H. Nature. 1995; 376: 660-669Crossref PubMed Scopus (1964) Google Scholar) have raised hopes that the long sought description of the proton translocation mechanism in transmembrane pumps might be near. According to the simplest version of the alternating access hypothesis (reviewed in Refs. 4Oesterhelt D. Tittor J. Bamberg E. J. Bioenerg. Biomembr. 1992; 24: 181-191Crossref PubMed Scopus (200) Google Scholar, 5Lanyi J.K. Nature. 1995; 375: 461-463Crossref PubMed Scopus (140) Google Scholar, 6Karlin A. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 5508-5509Crossref PubMed Scopus (3) Google Scholar), such transport is based on the energy-dependent cycling through two protein conformations in which the access of a single ion binding site changes from one membrane side to the other. It has been difficult to put this idea to a test because most proton pumps are large or multisubunit proteins in which the site of ion translocation is at some distance from the chemical reactions that drive it, and the chemical reactions are themselves complex. If there is a general mechanism in proton pumps, clues to it are more likely to come from a simpler system. Recent progress with bacteriorhodopsin, a small retinal protein in which the thermal reisomerization of photoisomerized retinal drives the proton transport, has yielded a step-by-step mechanism for the translocation cycle. The principles and perhaps some of the details in this mechanism, described briefly below, may prove to apply to other ion pumps. They seem relevant also to signal receptors (7Spudich J.L. Lanyi J.K. Curr. Opin. Cell Biol. 1996; 8: 452-457Crossref PubMed Scopus (58) Google Scholar). Bacteriorhodopsin is a seven-helix transmembrane protein, with an all-trans-retinal lying at a small angle to the membrane surface and linked via a protonated Schiff base to Lys216near the middle of helix G (Fig. 1). Most of the structure has been known at 3.5-Å resolution from two-dimensional crystals (8Grigorieff N. Ceska T.A. Downing K.H. Baldwin J.M. Henderson R. J. Mol. Biol. 1996; 259: 393-421Crossref PubMed Scopus (865) Google Scholar), and more recently the entire structure was described at 3.0 Å (9Kimura Y. Vassylyev D.G. Miyazawa A. Kidera A. Matsushima M. Mitsuoka K. Murata K. Hirai T. Fujiyoshi Y. Nature. 1997; 389: 206-211Crossref PubMed Scopus (410) Google Scholar). Higher resolution from three-dimensional crystals, at 2.5 Å is now available (10Peybay-Peyroula E. Rummel G. Rosenbusch J.P. Landau E.M. Science. 1997; 277: 1676-1681Crossref PubMed Scopus (820) Google Scholar). The interhelical cavity is divided by the Schiff base into extracellular and cytoplasmic “half-channels” that together describe the trajectory of the transported proton. The extracellular half-channel contains numerous charged or hydrogen-bonding residues, whereas the cytoplasmic region is simpler and mostly hydrophobic. In the first transport event after absorption of a photon, the Schiff base proton is mobilized by photoisomerization of the retinal to 13-cis,15-anti, and transferred to Asp85 in the extracellular region, causing the release of a proton to the surface. The Schiff base is then reprotonated from Asp96 from the cytoplasmic side. Asp96 in turn is reprotonated from the surface. These proton transfers together add up to translocation across the membrane. They and other reactions of the retinal and the protein during the cycle have been measured by various spectroscopic methods and consist of the interconversions of the intermediate states designated as J, K, L, M, N, and O, and substates of several of these. Much effort has been expended to describe these reactions and the protein residues involved (4Oesterhelt D. Tittor J. Bamberg E. J. Bioenerg. Biomembr. 1992; 24: 181-191Crossref PubMed Scopus (200) Google Scholar, 11Mathies R.A. Lin S.W. Ames J.B. Pollard W.T. Annu. Rev. Biophys. Biophys. Chem. 1991; 20: 491-518Crossref PubMed Scopus (504) Google Scholar, 12Rothschild K.J. J. Bioenerg. Biomembr. 1992; 24: 147-167Crossref PubMed Scopus (265) Google Scholar, 13Ebrey T.G. Jackson M. Thermodynamics of Membranes, Receptors and Channels. CRC Press, Inc., Boca Raton, FL1993: 353-387Google Scholar, 14Khorana H.G. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 1166-1171Crossref PubMed Scopus (110) Google Scholar, 15Lanyi J.K. Biochim. Biophys. Acta. 1993; 1183: 241-261Crossref PubMed Scopus (376) Google Scholar). But to understand bacteriorhodopsin as a proton pump we must know also what determines the rates of the proton transfers and how the pK a s and the geometry of the donors and acceptors change so as to give them a cytoplasmic-to-extracellular direction. Although the proton transport is the consequence of a “photocycle,” mechanistic clues have been gained recently from reactions of the unphotolyzed protein that correspond to various single photocycle steps. First, the observation of a biphasic titration curve for Asp85 suggested that its pK a is linked to the protonation of another residue (16Balashov S.P. Govindjee R. Imasheva E.S. Misra S. Ebrey T.G. Feng Y. Crouch R.K. Menick D.R. Biochemistry. 1995; 34: 8820-8834Crossref PubMed Scopus (132) Google Scholar, 17Balashov S.P. Imasheva E.S. Govindjee R. Ebrey T.G. Biophys. J. 1996; 70: 473-481Abstract Full Text PDF PubMed Scopus (194) Google Scholar) that turned out to be Glu204 (18Brown L.S. Sasaki J. Kandori H. Maeda A. Needleman R. Lanyi J.K. J. Biol. Chem. 1995; 270: 27122-27126Abstract Full Text Full Text PDF PubMed Scopus (278) Google Scholar, 19Richter H.T. Brown L.S. Needleman R. Lanyi J.K. Biochemistry. 1996; 35: 4054-4062Crossref PubMed Scopus (164) Google Scholar, 20Richter H.T. Needleman R. Lanyi J.K. Biophys. J. 1996; 71: 3392-3398Abstract Full Text PDF PubMed Scopus (29) Google Scholar) perhaps together with liganded water but certainly other residues near the extracellular surface, such as Glu194. The nature of the linkage is that protonation of Asp85 will cause deprotonation of the Glu204site and vice versa. This is the kind of coupling that would cause proton release at the extracellular surface after proton transfer from the Schiff base to Asp85 (21Heberle J. Dencher N.A. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 5996-6000Crossref PubMed Scopus (158) Google Scholar, 22Scherrer P. Alexiev U. Marti T. Khorana H.G. Heyn M.P. Biochemistry. 1994; 33: 13684-13692Crossref PubMed Scopus (69) Google Scholar, 23Alexiev U. Marti T. Heyn M.P. Khorana H.G. Scherrer P. Biochemistry. 1994; 33: 13693-13699Crossref PubMed Scopus (47) Google Scholar, 24Zimányi L. Váró G. Chang M. Ni B. Needleman R. Lanyi J.K. Biochemistry. 1992; 31: 8535-8543Crossref PubMed Scopus (204) Google Scholar, 25Cao Y. Brown L.S. Sasaki J. Maeda A. Needleman R. Lanyi J.K. Biophys. J. 1995; 68: 1518-1530Abstract Full Text PDF PubMed Scopus (79) Google Scholar) even though Asp85 itself remains protonated until the end of the photocycle (26Bousché O. Sonar S. Krebs M.P. Khorana H.G. Rothschild K.J. Photochem. Photobiol. 1992; 56: 1085-1095Crossref PubMed Scopus (71) Google Scholar, 27Souvignier G. Gerwert K. Biophys. J. 1992; 63: 1393-1405Abstract Full Text PDF PubMed Scopus (98) Google Scholar, 28Hessling B. Souvignier G. Gerwert K. Biophys. J. 1993; 65: 1929-1941Abstract Full Text PDF PubMed Scopus (169) Google Scholar). Conversely, once the proton is released at a pH higher than the pK a of the Glu204 site, the pK a for Asp85 will rise. Under physiological conditions, where the difference between the pH and the pK a for the release is large, the proton release will shift the protonation equilibrium between Asp85 and the Schiff base toward virtually complete and unidirectional proton transfer. The second observation, made from x-ray diffraction, was that deprotonation of the Schiff base of the D85N mutant by raising the pH in the dark caused the protein to assume an equilibrium mixture of conformations that exhibit structural changes seen otherwise only in the M photointermediate (29Kataoka M. Kamikubo H. Tokunaga F. Brown L.S. Yamazaki Y. Maeda A. Sheves M. Needleman R. Lanyi J.K. J. Mol. Biol. 1994; 243: 621-638Crossref PubMed Scopus (128) Google Scholar). In the D85N/D96N double mutant the equilibrium contained a large amount of the M-like conformation even with the Schiff base protonated. The pK a s for the protonated Schiff base and the changes in crystallographic parameters for D85N were the same, and the isomeric composition of the retinal was indifferent to the shift of protein structure (30Brown L.S. Kamikubo H. Zimányi L. Kataoka M. Tokunaga F. Verdegem P. Lugtenburg J. Lanyi J.K. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 5040-5044Crossref PubMed Scopus (70) Google Scholar). As expected from this, the pK a of the Schiff base in D85N was nearly unchanged when the retinal was replaced with an analogue locked in the all-trans configuration. These observations provided a hint to the cause of the proton transfer switch in the photocycle that allows reprotonation of the Schiff base from the cytoplasmic side; if it is a result of the shift of the global protein conformation, it depends on deprotonation of the Schiff base, i.e. loss of interaction between the protonated Schiff base and its complex counterion rather than directly on the isomeric state of the retinal. In the third study (31Richter H.T. Needleman R. Kandori H. Maeda A. Lanyi J.K. Biochemistry. 1996; 35: 15461-15466Crossref PubMed Scopus (55) Google Scholar), mutations in the extracellular proton channel were shown to cause parallel decreases in the rate of the final photocycle step and the deprotonation of Asp85 in pH jump experiments in the dark. The correlation of the rates over 3 orders of magnitude suggested that the loss of proton from Asp85 to the Glu204 site or directly to the medium depending on the conditions is the rate-limiting step in the recovery of the the also caused the of the but for these there was correlation with the deprotonation of In these a interaction between the or of the retinal and the protein with reisomerization to as suggested by other of J.K. U. S. Proc. Natl. Acad. Sci. U. S. A. 1995; PubMed Scopus Google Scholar, O. B. N. Sheves M. Lanyi J.K. Brown L.S. F. Biochemistry. 1996; 35: PubMed Scopus Google Scholar, S. D. Henderson R. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: PubMed Scopus Google Scholar). These some in the and to the proton transport mechanism that The site of the protonated Schiff base and the Asp85 and water A. Sasaki J. Y. T. Biochemistry. 1992; 31: PubMed Scopus Google Scholar, A. Sasaki J. Yamazaki Y. Needleman R. Lanyi J.K. Biochemistry. 1994; 33: PubMed Scopus Google Scholar, H. Yamazaki Y. Sasaki J. Needleman R. Lanyi J.K. Maeda A. J. Chem. 1995; Scopus Google Scholar, M. Sasaki J. Kandori H. Ebrey T.G. Needleman R. Lanyi J.K. Maeda A. Biochemistry. 1996; 35: PubMed Scopus Google Scholar, Sonar S. Marti T. Khorana H.G. Rothschild K.J. Biochemistry. 1994; 33: PubMed Scopus Google Scholar) that the of the retinal from all-trans to changes this geometry L.S. Y. Sheves M. Yamazaki Y. Maeda A. Needleman R. Lanyi J.K. Biochemistry. 1994; 33: PubMed Scopus Google Scholar, Y. Sheves M. J. Chem. 1993; Scopus Google Scholar) and the the retinal G. K. Chem. Scopus Google Scholar, K. F. P. J. Mol. Scopus Google Scholar), causing the pK a difference between the Schiff base and Asp85 to from to L.S. L. Needleman R. Lanyi J.K. Biophys. J. 1993; 65: Full Text PDF PubMed Scopus Google Scholar). the proton transfer in the and of the transport cycle that As Asp85 linkage of the pK a s of the Glu204 site (Fig. the pK a of the Glu204 site to from to As a Glu204 and the the proton to the surface. K. L. S. M. S. S. Y. R. and J. K. for pH release of the proton to the extracellular surface is This in turn the pK a of Asp85 to S.P. Imasheva E.S. Govindjee R. Ebrey T.G. Biophys. J. 1996; 70: 473-481Abstract Full Text PDF PubMed Scopus (194) Google Scholar, 19Richter H.T. Brown L.S. Needleman R. Lanyi J.K. Biochemistry. 1996; 35: 4054-4062Crossref PubMed Scopus (164) Google Scholar, Biophys. J. 1996; 70: Full Text PDF PubMed Scopus (55) Google Scholar), and its protonation equilibrium with the Schiff base toward nearly proton transfer L. Váró G. Chang M. Ni B. Needleman R. Lanyi J.K. Biochemistry. 1992; 31: 8535-8543Crossref PubMed Scopus (204) Google Scholar). This as the that was from the of changes G. Lanyi J.K. Biochemistry. 1991; PubMed Scopus (200) Google Scholar), the first of the transport cycle (Fig. of the Schiff base from Asp85 is now the proton transfer the charged Schiff complex to a of the structure of the protein is and an conformation is (30Brown L.S. Kamikubo H. Zimányi L. Kataoka M. Tokunaga F. Verdegem P. Lugtenburg J. Lanyi J.K. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 5040-5044Crossref PubMed Scopus (70) Google Scholar). from and of the M state at the cytoplasmic of and G N.A. D. G. G. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, Dencher N.A. D. G. G. J. 1991; PubMed Scopus Google Scholar, M. Kataoka M. Y. Tokunaga F. 1991; PubMed Scopus Google Scholar, H. Kataoka M. Váró G. T. Tokunaga F. Needleman R. Lanyi J.K. Proc. Natl. Acad. Sci. U. S. A. 1996; PubMed Scopus (110) Google Scholar, J. Biophys. J. 1994; Full Text PDF PubMed Scopus Google Scholar, S. M. D. Henderson R. J. 1993; PubMed Scopus Google Scholar, J. Biochemistry. 1996; 35: PubMed Scopus Google Scholar). shift of the absorption of the with Schiff base at this in the mutant but in the L. Y. Chang M. Ni B. Needleman R. Lanyi J.K. Photochem. Photobiol. 1992; 56: PubMed Scopus Google Scholar) suggested that the Schiff base into most likely through water (8Grigorieff N. Ceska T.A. Downing K.H. Baldwin J.M. Henderson R. J. Mol. Biol. 1996; 259: 393-421Crossref PubMed Scopus (865) Google Scholar, R. Baldwin J.M. Ceska T.A. F. E. Downing K.H. J. Mol. Biol. PubMed Scopus Google Scholar) with after proton transfer from Asp85 is made by the pK a of this the of Schiff base is to The of such an based on at the Schiff base, is suggested also by the observation that some conditions transport be even when Asp85 and Asp96 are replaced with residues J. U. D. Bamberg E. Biophys. J. 1994; Full Text PDF PubMed Scopus Google Scholar, U. Tittor J. Bamberg E. D. Biochemistry. 1997; PubMed Scopus Google Scholar). The change in geometry the second of proton translocation (Fig. this in the photocycle the pK a of the Schiff base is L.S. Lanyi J.K. Proc. Natl. Acad. Sci. U. S. A. 1996; PubMed Scopus Google Scholar), i.e. than its of S. M. A. J. Biochemistry. PubMed Scopus Google Scholar), but the pK a of Asp96 is to be at as high as as in the unphotolyzed protein S. D. P. Biophys. J. 1994; Full Text PDF PubMed Scopus Google Scholar). The pK a will to so as to Asp96 a proton Y. Váró G. Needleman R. Lanyi J.K. Biochemistry. 1993; PubMed Scopus Google Scholar). The to be a protein conformation change in the M intermediate when this state is in the mutant and that of the of the cytoplasmic end of helix N.A. D. G. G. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, Dencher N.A. D. G. G. J. 1991; PubMed Scopus Google Scholar, M. Kataoka M. Y. Tokunaga F. 1991; PubMed Scopus Google Scholar, H. Kataoka M. Váró G. T. Tokunaga F. Needleman R. Lanyi J.K. Proc. Natl. Acad. Sci. U. S. A. 1996; PubMed Scopus (110) Google Scholar, J. Biophys. J. 1994; Full Text PDF PubMed Scopus Google Scholar, S. M. D. Henderson R. J. 1993; PubMed Scopus Google Scholar, J. Biochemistry. 1996; 35: PubMed Scopus Google Scholar). The of by Y. Váró G. Chang M. Ni B. Needleman R. Lanyi J.K. Biochemistry. 1991; PubMed Scopus (194) Google Scholar) and G. Lanyi J.K. Biochemistry. 1995; 34: PubMed Scopus Google Scholar) suggested that it is the of the cytoplasmic region that the pK a of Asp96 and this residue a proton to the Schiff The of chemical of and at the cytoplasmic end of helix and the to the that is of this helix L.S. Váró G. Needleman R. Lanyi J.K. Biophys. J. 1995; Full Text PDF PubMed Scopus Google Scholar). of the of helix the high pK a of Asp96 L. Y. Needleman R. M. Lanyi J.K. Biochemistry. 1993; PubMed Scopus Google Scholar), and this residue is then reprotonated from the cytoplasmic surface. The of the of the protein in the during this M. Kataoka M. Y. Tokunaga F. 1991; PubMed Scopus Google Scholar) may be the cause of the in the two-dimensional of the membrane G. Needleman R. Lanyi J.K. Biophys. J. 1996; 70: Full Text PDF PubMed Scopus Google Scholar). of the retinal to a shown in is by proton transfer from Asp85 to the Glu204 site and of the retinal (31Richter H.T. Needleman R. Kandori H. Maeda A. Lanyi J.K. Biochemistry. 1996; 35: 15461-15466Crossref PubMed Scopus (55) Google Scholar), the the recovery of the proton of a and the Glu204 site a at the end of the photocycle is the for the of this It is because the pK a of Asp85 at this to its that proton transport a high transmembrane proton and is to drive the of H. D. Biochemistry. PubMed Scopus Google Scholar, A. Biochim. Biophys. Acta. PubMed Scopus Google Scholar). Much that there is water near the Schiff base and and its changes the near the Schiff base and Asp85 was suggested to the proton transfer Y. Sheves M. J. Chem. 1993; Scopus Google Scholar, N. Sheves M. M. Biochemistry. 1995; 34: PubMed Scopus Google Scholar). water that Asp85 to Glu204 in structures from K. Sheves M. Biochemistry. 1994; 33: PubMed Scopus Google Scholar, C. J. J. Chem. 1995; Scopus Google Scholar) may the H.T. Brown L.S. Needleman R. Lanyi J.K. Biochemistry. 1996; 35: 4054-4062Crossref PubMed Scopus (164) Google Scholar) in the linked pK a s of Asp85 and the The access of the Schiff base to Asp96 has been suggested to be through water at the site D. Sheves M. K. Biophys. J. 1995; Full Text PDF PubMed Scopus Google Scholar). between the Schiff base and Asp96 were to proton transfer across the distance in the of the protein (8Grigorieff N. Ceska T.A. Downing K.H. Baldwin J.M. Henderson R. J. Mol. Biol. 1996; 259: 393-421Crossref PubMed Scopus (865) Google Scholar, Y. Vassylyev D.G. Miyazawa A. Kidera A. Matsushima M. Mitsuoka K. Murata K. Hirai T. Fujiyoshi Y. Nature. 1997; 389: 206-211Crossref PubMed Scopus (410) Google Scholar). The of on this Y. Váró G. Chang M. Ni B. Needleman R. Lanyi J.K. Biochemistry. 1991; PubMed Scopus (194) Google Scholar) suggested that first an then a amount of water near with the and the of a at the cytoplasmic surface is what the proton of this residue as a proton to the Schiff base and then an from the cytoplasmic surface. in the step of the proton is from Asp85 to Glu204 most likely through the of the water that the large with proton release and the deprotonation of Asp85 that (31Richter H.T. Needleman R. Kandori H. Maeda A. Lanyi J.K. Biochemistry. 1996; 35: 15461-15466Crossref PubMed Scopus (55) Google Scholar) that water an in these The Schiff base and its to the single site in this proton The of the of the protein may be to to and a proton from this If this is we between an site for the and ion transfer and a that to it by its access to membrane surface. is for this from an change in the ion of Asp85 with a the residue in the pump P. A. H. F. D. J. PubMed Scopus Google Scholar), bacteriorhodopsin the spectroscopic and the transport in the from the proton of J. Brown L.S. Kandori H. Maeda A. Needleman R. Lanyi J.K. Science. 1995; 269: PubMed Scopus Google Scholar). Although the transport mechanism in this bacteriorhodopsin and in G. Zimányi L. L. Needleman R. Lanyi J.K. Biophys. J. 1995; 68: Full Text PDF PubMed Scopus Google Scholar, G. Brown L.S. Sasaki J. Maeda A. Needleman R. Lanyi J.K. Biochemistry. 1995; 34: PubMed Scopus Google Scholar, G. Needleman R. Lanyi J.K. Biochemistry. 1995; 34: PubMed Scopus (79) Google Scholar, D. J. Chem. 1995; 35: Scopus Google Scholar) is it must binding of at the site and its translocation to the cytoplasmic the alternating of access in this protein the of only but also in be made to transport This will in a in which the and protonated all-trans-retinal Schiff are with an protonation and reprotonation that in translocation of E. Tittor J. D. Proc. Natl. Acad. Sci. U. S. A. 1993; Scopus Google Scholar). to the change of of bacteriorhodopsin, also a proton pump when an proton the a site near the Schiff base where otherwise the transported is G. Brown L.S. Needleman R. Lanyi J.K. Biochemistry. 1996; 35: PubMed Scopus Google Scholar). when are made for a proton to the Schiff base, the access change and the for will also The hypothesis that the of bacteriorhodopsin depends on proton to and from the surface than on the proton transfer at the site is by the that of the of mutations K.J. J. Bioenerg. Biomembr. 1992; 24: 147-167Crossref PubMed Scopus (265) Google Scholar, 14Khorana H.G. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 1166-1171Crossref PubMed Scopus (110) Google Scholar, 15Lanyi J.K. Biochim. Biophys. Acta. 1993; 1183: 241-261Crossref PubMed Scopus (376) Google Scholar) transport that have the proton transfer from the Schiff base to by the with a residue or by it protonated. even in the where transport of the kind in the is proton transport is some conditions J. U. D. Bamberg E. Biophys. J. 1994; Full Text PDF PubMed Scopus Google U. Tittor J. Bamberg E. D. Biochemistry. 1997; PubMed Scopus Google Scholar). other mutations that are are also of In the proton release to the extracellular surface protonation of Asp85 is Although proton release is until the end of the photocycle when the pK a of Asp85 is the of the proton transfers is (18Brown L.S. Sasaki J. Kandori H. Maeda A. Needleman R. Lanyi J.K. J. Biol. Chem. 1995; 270: 27122-27126Abstract Full Text Full Text PDF PubMed Scopus (278) Google Scholar, R. Misra S. S.P. Ebrey T.G. Crouch R.K. Menick D.R. Biophys. J. 1996; 71: Full Text PDF PubMed Scopus Google Scholar). In the reprotonation of the Schiff base and the proton from the cytoplasmic surface are of the Schiff base is directly from the surface but with the Y. Váró G. Needleman R. Lanyi J.K. Biochemistry. 1993; PubMed Scopus Google Scholar, H. Marti T. M. T. M. Khorana H.G. Heyn M.P. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, A. D. Biochim. Biophys. Acta. Scopus Google Scholar). the proton to membrane be loss of transport the other with these the rate of the and the rate of proton transport at high will be physiological pH the of the photocycle in to orders of as the rate-limiting step the of a proton at the cytoplasmic surface Y. Váró G. Chang M. Ni B. Needleman R. Lanyi J.K. Biochemistry. 1991; PubMed Scopus (194) Google Scholar). of residues at the cytoplasmic surface, such as that might also have in proton a of the photocycle J. D. Dencher N.A. J. Biochemistry. 1996; 35: PubMed Scopus Google Scholar). at the extracellular surface, the of the by orders of as the loss of the proton of Asp85 rate-limiting (18Brown L.S. Sasaki J. Kandori H. Maeda A. Needleman R. Lanyi J.K. J. Biol. Chem. 1995; 270: 27122-27126Abstract Full Text Full Text PDF PubMed Scopus (278) Google Scholar, H.T. Needleman R. Kandori H. Maeda A. Lanyi J.K. Biochemistry. 1996; 35: 15461-15466Crossref PubMed Scopus (55) Google Scholar). such must be the for the of the proton transfer described the of residues in the structure that may for release and of at the two (9Kimura Y. Vassylyev D.G. Miyazawa A. Kidera A. Matsushima M. Mitsuoka K. Murata K. Hirai T. Fujiyoshi Y. Nature. 1997; 389: 206-211Crossref PubMed Scopus (410) Google Scholar). the transport in bacteriorhodopsin is by an alternating access The change of the access of the proton binding site in steps. In the first and the of the transfer of proton between the Schiff base and the two membrane is by the magnitude and of a a to proton and Although this would by the of the transport, there may be in a change of the geometry to switch the of the Schiff The for other pumps may be that the ion transfer which the transported ion to the site and it, are more and more than the between the binding site and the two membrane