pH-dependent photoisomerization of retinal in proteorhodopsin

The early steps in the photocycle of the bacterial proton pump proteorhodopsin (PR) were analyzed by ultrafast pump/probe spectroscopy to compare the rate of retinal isomerization at alkaline and acidic pH values. At pH 9, the functionally important primary proton acceptor (Asp97, pK(a) = 7.7) is negatively charged; consequently, a reaction cycle analogous to the archaeal bacteriorhodopsin (BR) is observed. The excited electronic state of PR displays a pronounced biphasic decay with time constants of 400 fs and 8 ps. At pH 6 where Asp97 is protonated a similar biphasic decay is observed, although it is significantly slower (700 fs and 15 ps). The results indicate, in agreement to similar findings in other retinal proteins, that also in PR the charge distribution within the chromophore binding pocket is a major determinant for the rate and the efficiency of the primary reaction.     

Proteorhodopsin is a light-driven proton pump with variable vectoriality

Proteorhodopsin, a homologue of archaeal bacteriorhodopsin (BR), belongs to a newly identified family of retinal proteins from marine bacteria, which could play an important role in the energy balance of the biosphere. We cloned the cDNA sequence of proteorhodopsin by chemical gene synthesis, expressed the protein in Escherichia coli cells, purified and reconstituted the protein in its functional active state. The photocycle characteristics were determined by time-resolved absorption and Fourier transform infrared (FT-IR) spectroscopy. The pH-dependence of the absorption spectrum indicates that the pK(a) of the primary acceptor of the Schiff base proton (Asp97) is 7.68. Generally, the photocycle of proteorhodopsin is similar to that of BR, although an L-like photocycle intermediate was not detectable. Whereas at pH>7 an M-like intermediate is formed upon illumination, at pH 5 no M-like intermediate could be detected. As the photocycle kinetics do not change between the acidic and alkaline state of proteorhodopsin, the only difference between these two forms is the protonation status of Asp97. This is corroborated by time-resolved FT-IR spectroscopy, which demonstrates that proton transfer from the retinal Schiff base to Asp97 is observed at alkaline pH, but the other vibrational changes are essentially pH-independent.After reconstitution into proteoliposomes, light-induced proton currents of proteorhodopsin were measured in a compound membrane system where proteoliposomes were adsorbed to planar lipid bilayers. Our results show that proteorhodopsin is a light-driven proton pump with characteristics similar to those of BR at alkaline pH. However, at acidic pH, the direction of proton pumping is inverted. Complementary experiments were carried out on proteorhodopsin expressed heterologously in Xenopus laevis oocytes under voltage clamp conditions.The following results were obtained. (1) At alkaline pH, proteorhodopsin mediates outwardly directed proton pumping like BR. (2) The direction of proton pumping can be inverted, when Asp97 is protonated. (3) The current can be inverted by changes of the polarity of the applied voltage. (4) The light intensity-dependence of the photocurrents leads to the conclusion that the alkaline form of proteorhodopsin shows efficient proton pumping after sequential excitation by two photons.     

Initial reaction dynamics of proteorhodopsin observed by femtosecond infrared and visible spectroscopy

We present a comparative study using femtosecond pump/probe spectroscopy in the visible and infrared of the early photodynamics of solubilized proteorhodopsin (green absorbing variant) in D₂O with deprotonated (pD 9.2) and protonated (pD 6.4) primary proton acceptor Asp-97. The vis-pump/vis-probe experiments show a kinetic isotope effect that is more pronounced for alkaline conditions, thus decreasing the previously reported pH-dependence of the primary reaction of proteorhodopsin in H₂O. This points to a pH dependent H-bonding network in the binding pocket of proteorhodopsin, that directly influences the primary photo-induced dynamics. The vis-pump/IR-probe experiments were carried out in two different spectral regions and allowed to monitor the retinal CC (1500 cm⁻¹-1580 cm⁻¹) and CN stretching vibration as well as the amide I mode of the protein (1590 cm⁻¹-1680 cm⁻¹). Like the FTIR spectra of the K intermediate (PR_K-PR difference spectra) in this spectral range, the kinetic parameters and also the quantum efficiency of photo-intermediate formation are found to be virtually independent of the pD value.     

Proton transport by proteorhodopsin requires that the retinal Schiff base counterion Asp-97 be anionic

At pH >7, proteorhodopsin functions as an outward-directed proton pump in cell membranes, and Asp-97 and Glu-108, the homologues of the Asp-85 and Asp-96 in bacteriorhodopsin, are the proton acceptor and donor to the retinal Schiff base, respectively. It was reported, however [Friedrich, T. et al. (2002) J. Mol. Biol., 321, 821-838], that proteorhodopsin transports protons also at pH <7 where Asp-97 is protonated and in the direction reverse from that at higher pH. To explore the roles of Asp-97 and Glu-108 in the proposed pumping with variable vectoriality, we compared the photocycles of D97N and E108Q mutants, and the effects of azide on the photocycle of the E108Q mutant, at low and high pH. Unlike at high pH, at a pH low enough to protonate Asp-97 neither the mutations nor the effects of azide revealed evidence for the participation of the acidic residues in proton transfer, and as in the photocycle of the wild-type protein, no intermediate with unprotonated Schiff base accumulated. In view of these findings, and the doubts raised by absence of charge transfer after flash excitation at low pH, we revisited the question whether transport occurs at all under these conditions. In both oriented membrane fragments and liposomes reconstituted with proteorhodopsin, we found transport at high pH but not at low pH. Instead, proton transport activity followed the titration curve for Asp-97, with an apparent pK(a) of 7.1, and became zero at the pH where Asp-97 is fully protonated.     

The photochemical reaction cycle of proteorhodopsin at low pH

The proton acceptor group in the recently described retinal protein, proteorhodopsin has an unusually high pK(a) of 7.1. It was shown that at pH above this pK(a), illumination initiates a photocycle similar to that of bacteriorhodopsin, and the protein transports proton across the cell membrane. Recently it was reported that proteorhodopsin, unlike bacteriorhodopsin, transports protons at pH below the pK(a) of the proton acceptor, and this transport is in the reverse direction. We have investigated the photocycle of proteorhodopsin at such low pH. At pH 5, three spectrally distinct intermediates K, L, and N, and another spectrally silent one, PR', could be identified, but a deprotonated Schiff base containing M-like intermediate, characteristic for proton pumping activity, does not accumulate. All the reactions between the intermediates are close to equilibrium, except the last transition from PR' to PR, when the protein returns to its initial unexcited state in a quasiunidirectional reaction. The electric signal measurements indicate that although charge motions are detected inside the protein, their net dislocation is zero, indicating that contrary to the earlier reported, at low pH no charged particle is transported across the membrane.     

Ultrafast excited state proton transfer dynamics of 1,2-dihydroquinolines in methanol solution.

Femtosecond and picosecond dynamics of 2,2,4,6-tetramethyl-1,2-dihydroquinoline (1) and 1,2,2,4,6-pentamethyl-1,2-dihydroquinoline (2) were studied in MeOH, MeOD, and Pr(i)OH to probe the early events of the photoinduced proton transfer (PT) between 1,2-dihydroquinolines (DHQ) and a solvent. From studies in the two solvents MeOH and Pr(i)OH and by examining the effect of deuterium replacement of proton, it has been established that PT takes 150-200 fs in MeOH, but does not occur in Pr(i)OH. The formation of PT products in the ground state proceeds concurrently to the relaxation of the higher vibrational excited singlet state to the thermally equilibrated state S(1) of DHQ. The absorption spectrum of the S(1) state was registered, and the time constant of its decay in MeOH (ca. 1 ns) agrees well with the lifetime of fluorescence measured recently by single photon counting.     

Proton release and uptake of pharaonis phoborhodopsin (sensory rhodopsin II) reconstituted into phospholipids

pharaonis phoborhodopsin (ppR, also called pharaonis sensory rhodopsin II, psRII) is a photo-receptor for negative phototaxis in Natronobacterium pharaonis. During the photoreaction cycle (photocycle), ppR exhibits intraprotein proton movements, resulting in proton pumping from the cytoplasmic to the extracellular side, although it is weak. In this study, light-induced proton uptake and release of ppR reconstituted with phospholipid were analyzed using a SnO(2) electrode. The reconstituted ppR exhibited properties in proton uptake and release that are different from those of dodecyl maltoside solubilized samples. It showed fast proton release before the decay of ppR(M) (M-photointermediate) followed by proton uptake, which was similar to that of bacteriorhodopsin (BR), a light-driven proton pump. Mutant analysis assigned Asp193 to one (major) of the members of the proton-releasing group (PRG). Fast proton release was observed only when the pH was approximately 5-8 in the presence of Cl(-). When Cl(-) was replaced with SO(4)(2-), the reconstituted ppR did not exhibit fast proton release at any pH, suggesting Cl(-) binding around PRG. PRG in BR consists of Glu204 (Asp193 in ppR) and Glu194 (Pro183 in ppR). Replacement of Pro183 by Glu/Asp, a negatively charged residue, led to Cl(-)-independent fast proton release. The transducer binding affected the properties of PRG in ppR in the ground state and in the ppR(M) state, suggesting that interaction with the transducer extends to the extracellular surface of ppR. Differences and similarities in the molecular mechanism of the proton movement between ppR and BR are discussed.     

Characterisation of Schiff base and chromophore in green proteorhodopsin by solid-state NMR

The proteorhodopsin family consists of hundreds of homologous retinal containing membrane proteins found in bacteria in the photic zone of the oceans. They are colour tuned to their environment and act as light-driven proton pumps with a potential energetic and regulatory function. Precise structural details are still unknown. Here, the green proteorhodopsin variant has been selected for a chemical shift analysis of retinal and Schiff base by solid-state NMR. Our data show that the chromophore exists in mainly all-trans configuration in the proteorhodopsin ground state. The optical absorption maximum together with retinal and Schiff base chemical shifts indicate a strong interaction network between chromophore and opsin. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. Mark Lorch and Andreas C. Woerner contributed equally to this work.     

Ultrafast excited state dynamics of the protonated Schiff base of all-trans retinal in solvents

We present a comparative study of the ultrafast photophysics of all-trans retinal in the protonated Schiff base form in solvents with different polarities and viscosities. Steady-state spectra of retinal in the protonated Schiff base form show large absorption-emission Stokes shifts (6500-8100 cm(-1)) for both polar and nonpolar solvents. Using a broadband fluorescence up-conversion experiment, the relaxation kinetics of fluorescence is investigated with 120 fs time resolution. The time-zero spectra already exhibit a Stokes-shift of approximately 6000 cm(-1), indicating depopulation of the Franck-Condon region in < or =100 fs. We attribute it to relaxation along skeletal stretching. A dramatic spectral narrowing is observed on a 150 fs timescale, which we assign to relaxation from the S(2) to the S(1) state. Along with the direct excitation of S(1), this relaxation populates different quasistationary states in S(1), as suggested from the existence of three distinct fluorescence decay times with different decay associated spectra. A 0.5-0.65 ps decay component is observed, which may reflect the direct repopulation of the ground state, in line with the small isomerization yield in solvents. Two longer decay components are observed and are attributed to torsional motion leading to photo-isomerization. The various decay channels show little or no dependence with respect to the viscosity or dielectric constant of the solvents. This suggests that in the protein, the bond selectivity of isomerization is mainly governed by steric effects.     

The Photocycle and Proton Translocation Pathway in a Cyanobacterial Ion-Pumping Rhodopsin

The genome of thylakoidless cyanobacterium Gloeobacter violaceus encodes a fast-cycling rhodopsin capable of light-driven proton transport. We characterize the dark state, the photocycle, and the proton translocation pathway of GR spectroscopically. The dark state of GR contains predominantly all-trans-retinal and, similar to proteorhodopsin, does not show the light/dark adaptation. We found an unusually strong coupling between the conformation of the retinal and the site of Glu¹³², the homolog of Asp⁹⁶ of BR. Although the photocycle of GR is similar to that of proteorhodopsin in general, it differs in accumulating two intermediates typical for BR, the L-like and the N-like states. The latter state has a deprotonated cytoplasmic proton donor and is spectrally distinct from the strongly red-shifted N intermediate known for proteorhodopsin. The proton uptake precedes the release and occurs during the transition to the O intermediate. The proton translocation pathway of GR is similar to those of other proton-pumping rhodopsins, involving homologs of BR Schiff base proton acceptor and donor Asp⁸⁵ and Asp⁹⁶ (Asp¹²¹ and Glu¹³²). We assigned a pair of FTIR bands (positive at 1749 cm⁻¹ and negative at 1734 cm⁻¹) to the protonation and deprotonation, respectively, of these carboxylic acids.     

Different structural changes occur in blue- and green-proteorhodopsins during the primary photoreaction

We examine the structural changes during the primary photoreaction in blue-absorbing proteorhodopsin (BPR), a light-driven retinylidene proton pump, using low-temperature FTIR difference spectroscopy. Comparison of the light-induced BPR difference spectrum recorded at 80 K to that of green-absorbing proteorhodopsin (GPR) reveals that there are several differences in the BPR and GPR primary photoreactions despite the similar structure of the retinal chromophore and all-trans --> 13-cis isomerization. Strong bands near 1700 cm(-1) assigned previously to a change in hydrogen bonding of Asn230 in GPR are still present in BPR. However, additional bands in the same region are assigned on the basis of site-directed mutagenesis to changes occurring in Gln105. In the amide II region, bands are assigned on the basis of total (15)N labeling to structural changes of the protein backbone, although no such bands were previously observed for GPR. A band at 3642 cm(-1) in BPR, assigned to the OH stretching mode of a water molecule on the basis of H2(18)O substitution, appears at a different frequency than a band at 3626 cm(-1) previously assigned to a water molecule in GPR. However, the substitution of Gln105 for Leu105 in BPR leads to the appearance of both bands at 3642 and 3626 cm(-1), indicating the waters assigned in BPR and GPR exist in separate distinct locations and can coexist in the GPR-like Q105L mutant of BPR. These results indicate that there exist significant differences in the conformational changes occurring in these two types proteorhodopsin during the initial photoreaction despite their similar chromophore structures, which might reflect a different arrangement of water in the active site as well as substitution of a hydrophilic for hydrophobic residue at residue 105.     

Characterization of the chimeric seven-transmembrane protein containing conserved region of helix C–F of microbial rhodopsin from Ganges River

Proteorhodopsin (PR) is a light-driven proton pump that has been found in a variety of marine bacteria. Recently, many PR-like genes were found in non-marine environments. The goal of this study is to explore the function of rhodopsins that exist only as partial proteo-opsin genes using chimeras with marine green PR (GPR). We isolated nine partial genes of PR homologues using polymerase chain reaction (PCR) and chose three homologues of GPR from the surface of the Ganges River, which has earned them the name “CFR, Chimeric Freshwater Rhodopsin.” In order to characterize the proteins, we constructed the cassette based on GPR sequence without helices C to F and inserted the isolated conserved partial sequences. When expressed in E. coli, we could observe light-driven proton pumping activity similar to proteorhodopsin, however, photocycle kinetics of CFRs are much slower than proteorhodopsin. Half-time decay of O intermediates of CFRs ranged between 143 and 333 ms at pH 10; their absorption maxima were between 515 and 522 nm at pH 7. We can guess that the function of native rhodopsin, a retinal protein of fresh water bacteria, may be a light-driven proton transport based on the results from chimeric freshwater rhodopsins. This approach will enable many labs that keep reporting partial PCR-based opsin sequences to finally characterize their proteins.     

Reconstituted energy transfer from antenna pigment-protein to reaction centres isolated from Rhodopseudomonas sphaeroides.

Efficient energy transfer has been reconstituted between an antenna pigment-protein and reaction centres isolated from the photosynthetic membrane of Rhodopseudomonas sphaeroides. The reconstituted system has fluorescence induction kinetics and fluorescence yields similar to those obtained from antenna bacteriochlorophyll in chromatophores. The results indicated that closed reaction centres quench fluorescence from the antenna pigment-protein, although not as strongly as photochemically active reaction centres. The measurement of fluorescence yields from chromatophores of the reaction centreless mutant PM-8 and of the parent strain Ga confirmed these observations. The fluorescence yield from the reconstituted system was approximately the same whether the reaction centres had been closed by photo-oxidation of the bacteriochlorophyll electron donor or chemical reduction of the primary acceptor, indicating a similar lifetime for the excited singlet state in both states of the reaction centres.     

Reconstituted energy transfer from antenna pigment-protein to reaction centres isolated from Rhodopseudomonas sphaeroides

Efficient energy transfer has been reconstituted between an antenna pigment-protein and reaction centres isolated from the photosynthetic membrane of Rhodopseudomonas sphaeroides. The reconstituted system has fluorescence induction kinetics and fluorescence yields similar to those obtained from antenna bacteriochlorophyll in chromatophores. The results indicated that closed reaction centres quench fluorescence from the antenna pigment-protein, although not as strongly as photochemically active reaction centres. The measurement of fluorescence yields from chromatophores of the reaction centreless mutant PM-8 and of the parent strain Ga confirmed these observations.The fluorescence yield from the reconstituted system was approximately the same whether the reaction centres had been closed by photo-oxidation of the bacteriochlorophyll electron donor or chemical reduction of the primary acceptor, indicating a similar lifetime for the excited singlet state in both states of the reaction centres.     

Proton transfers in the photochemical reaction cycle of proteorhodopsin

The spectral and photochemical properties of proteorhodopsin (PR) were determined to compare its proton transport steps to those of bacteriorhodopsin (BR). Static and time-resolved measurements on wild-type PR and several mutants were done in the visible and infrared (FTIR and FT-Raman). Assignment of the observed C=O stretch bands indicated that Asp-97 and Glu-108 serve as the proton acceptor and donor, respectively, to the retinal Schiff base, as do the residues at corresponding positions in BR, but there are numerous spectral and kinetic differences between the two proteins. There is no detectable dark-adaptation in PR, and the chromophore contains nearly entirely all-trans retinal. Because the pK(a) of Asp-97 is relatively high (7.1), the proton-transporting photocycle is produced only at alkaline pH. It contains at least seven transient states with decay times in the range from 10 micros to 200 ms, but the analysis reveals only three distinct spectral forms. The first is a red-shifted K-like state. Proton release does not occur during the very slow (several milliseconds) rise of the second, M-like, intermediate, consistent with lack of the residues facilitating extracellular proton release in BR. Proton uptake from the bulk, presumably on the cytoplasmic side, takes place prior to release (tau approximately 2 ms), and coincident with reprotonation of the retinal Schiff base. The intermediate produced by this process contains 13-cis retinal as does the N state of BR, but its absorption maximum is red-shifted relative to PR (like the O state of BR). The decay of this N-like state is coupled to reisomerization of the retinal to all-trans, and produces a state that is O-like in its C-C stretch bands, but has an absorption maximum apparently close to that of unphotolyzed PR.     

Some properties of membrane-bound, solubilized and reconstituted into liposomes H+-ATPase of vacuoles of Saccharomyces carlsbergensis

Vacuoles of yeast grown in peptone medium possessed high ATPase activity (up to 1 mumol X mg protein-1 X min-1). Membrane-bound and solubilized ATPase activities were insensitive to vanadate and azide, but were inhibited by NO-3 . K+ and cyclic AMP stimulated both membrane-bound and solubilized ATPase activities. Dio-9 activated the membrane form of vacuolar ATPase 1.5-2-fold and did not affect the solubilized enzyme. Solubilized and partially purified vacuolar ATPase was reconstituted with soy-bean phospholipids by a freeze-thaw procedure. ATPase activities in native vacuoles and proteoliposomes were stimulated effectively by Dio-9, the protonophore FCCP and ionophores valinomycin and nigericin. ATP-dependent H+ transport into proteoliposomes was also shown by quenching of ACMA fluorescence. Vacuolar and partially purified ATPase preparations possessed also GTPase activity. Unlike ATPase, however, GTPase was not incorporated as a proton pump into liposomes.     

Aspartate-histidine interaction in the retinal schiff base counterion of the light-driven proton pump of Exiguobacterium sibiricum

One of the distinctive features of eubacterial retinal-based proton pumps, proteorhodopsins, xanthorhodopsin, and others, is hydrogen bonding of the key aspartate residue, the counterion to the retinal Schiff base, to a histidine. We describe properties of the recently found eubacterium proton pump from Exiguobacterium sibiricum (named ESR) expressed in Escherichia coli, especially features that depend on Asp-His interaction, the protonation state of the key aspartate, Asp85, and its ability to accept a proton from the Schiff base during the photocycle. Proton pumping by liposomes and E. coli cells containing ESR occurs in a broad pH range above pH 4.5. Large light-induced pH changes indicate that ESR is a potent proton pump. Replacement of His57 with methionine or asparagine strongly affects the pH-dependent properties of ESR. In the H57M mutant, a dramatic decrease in the quantum yield of chromophore fluorescence emission and a 45 nm blue shift of the absorption maximum with an increase in the pH from 5 to 8 indicate deprotonation of the counterion with a pK(a) of 6.3, which is also the pK(a) at which the M intermediate is observed in the photocycle of the protein solubilized in detergent [dodecyl maltoside (DDM)]. This is in contrast with the case for the wild-type protein, for which the same experiments show that the major fraction of Asp85 is deprotonated at pH >3 and that it protonates only at low pH, with a pK(a) of 2.3. The M intermediate in the wild-type photocycle accumulates only at high pH, with an apparent pK(a) of 9, via deprotonation of a residue interacting with Asp85, presumably His57. In liposomes reconstituted with ESR, the pK(a) values for M formation and spectral shifts are 2-3 pH units lower than in DDM. The distinctively different pH dependencies of the protonation of Asp85 and the accumulation of the M intermediate in the wild-type protein versus the H57M mutant indicate that there is strong Asp-His interaction, which substantially lowers the pK(a) of Asp85 by stabilizing its deprotonated state.     

Critical role of asp227 in the photocycle of proteorhodopsin

The photocycle of the proton acceptor complex mutant D227N of the bacterial retinal protein proteorhodopsin is investigated employing steady state pH-titration experiments in the UV-visible range as well as femtosecond-pump-probe spectroscopy and flash photolysis in the visible spectral range. The evaluation of the pH-dependent spectra showed that the neutralization of the charge at position 227 has a remarkable influence on the ground state properties of the protein. Both the pK(a) values of the primary proton acceptor and of the Schiff base are considerably decreased. Femtosecond-time-resolved measurements demonstrate that the general S(1) deactivation pathway; that is, the K-state formation is preserved in the D227N mutant. However, the pH-dependence of the reaction rate is lost by the substitution of Asp227 with an asparagine. Also no significant kinetic differences are observed upon deuteration. This is explained by the lack of a strongly hydrogen-bonded water in the vicinity of Asp97, Asp227, and the Schiff base or a change in the hydrogen bonding of it (Ikeda et al. (2007) Biochemistry46, 5365-5373). The flash photolysis measurements prove a considerably elongated photocycle with pronounced pH-dependence. Interestingly, at pH 9 the M-state is visible until the end of the reaction cycle, leading to the conclusion that the mutation does not only lower the pK(a) of the Schiff base in the unphotolyzed ground state but also prevents an efficient reprotonation reaction.     

The chromophore retinal hinders passive proton/hydroxide ion translocation through bacteriorhodopsin

Experiments have been performed to examine any influence of the chromophore retinal in bacteriorhodopsin (BR) on the passive proton/hydroxide ion flux through this integral membrane protein. BR was reconstituted into dimyristoylphosphatidylcholine (DMPC)-phosphatidylserine or DMPC-dimyristoylphosphatidylglycerol unilamellar vesicles with molar lipid to protein ratios ranging from 30 to 150. The entrapped fluorescence dye pyranine served as a reliable indicator of the internal proton concentration. Transmembrane pH-gradients were quickly established across the vesicular membrane and the kinetics of the induced fluorescence changes were compared for vesicles with incorporated native BR, BR bleached to the chromophore-free protein bacterioopsin, and BR regenerated from bacterioopsin with all-trans-retinal, respectively. For aggregated protein molecules, the H+/OH- diffusion across bacterioopsin was always considerably faster than that through the protein containing covalently bound retinal. The decay rate of the imposed pH-gradient was 4.4-9.1 and 2.0-5.1 times slower for native and regenerated BR, respectively, as compared to bacterioopsin. Stepwise regeneration of bacterioopsin with all-trans-retinal revealed a linear dependence of the predominant delta pH-decay time on the degree of regeneration. Essentially the same observations were made with monomeric protein molecules in vesicular lipid membranes. The results demonstrate that the chromophore retinal itself blocks the H+/OH- conducting pathway across the transmembrane protein BR or indirectly controls this path by inducing conformational changes in the protein upon binding.     

Isolation and reconstitution of the clathrin-coated vesicle proton translocating complex

Clathrin-coated vesicles contain a proton translocating ATPase which is insensitive to azide but inhibited by N-ethylmaleimide. The ATP hydrolytic subunit of this proton pump has been solubilized, partially purified, and reconstituted into H+-ATPase-depleted coated vesicle membranes (Xie, X.-S., Stone, D.K., and Racker, E. (1984) J. Biol. Chem. 259, 11676-11678). In this communication we report that the entire proton transporting complex has been solubilized and purified 200-fold. The complex, when reconstituted into brain lipid liposomes, catalyzes azide-resistant, N-ethylmaleimide-sensitive H+ transport manifested as both generation of a pH gradient and an electrical gradient. The complex has an apparent molecular mass of 530 kDa.