Spectroscopic and Photochemical Characterization of a Deep Ocean Proteorhodopsin

A second group of proteorhodopsin-encoding genes (blue-absorbing proteorhodopsin, BPR) differing by 20–30% in predicted primary structure from the first-discovered green-absorbing (GPR) group has been detected in picoplankton from Hawaiian deep sea water. Here we compare BPR and GPR absorption spectra, photochemical reactions, and proton transport activity. The photochemical reaction cycle of Hawaiian deep ocean BPR in cells is 10-fold slower than that of GPR with very low accumulation of a deprotonated Schiff base intermediate in cells and exhibits mechanistic differences, some of which are due to its glutamine residue rather than leucine at position 105. In contrast to GPR and other characterized microbial rhodopsins, spectral titrations of BPR indicate that a second titratable group, in addition to the retinylidene Schiff base counterion Asp-97, modulates the absorption spectrum near neutral pH. Mutant analysis confirms that Asp-97 and Glu-108 are proton acceptor and proton donor, respectively, in retinylidene Schiff base proton transfer reactions during the BPR photocycle as previously shown for GPR, but BPR contains an alternative acceptor evident in its D97N mutant, possibly the same as the second titratable group modulating the absorption spectrum. BPR, similar to GPR, carries out outward light-driven proton transport in Escherichia coli vesicles but with a reduced translocation rate attributable to its slower photocycle. In energized E. coli cells at physiological pH, the net effect of BPR photocycling is to generate proton currents dominated by a triggered proton influx, rather than efflux as observed with GPR-containing cells. Reversal of the proton current with the K+-ionophore valinomycin supports that the influx is because of voltagegated channels in the E. coli cell membrane. These observations demonstrate diversity in photochemistry and mechanism among proteorhodopsins. Calculations of photon fluence rates at different ocean depths show that the difference in photocycle rates between GPR and BPR as well as their different absorption maxima may be explained as an adaptation to the different light intensities available in their respective marine environments. Finally, the results raise the possibility of regulatory (i.e. sensory) rather than energy harvesting functions of some members of the proteorhodopsin family. A second group of proteorhodopsin-encoding genes (blue-absorbing proteorhodopsin, BPR) differing by 20–30% in predicted primary structure from the first-discovered green-absorbing (GPR) group has been detected in picoplankton from Hawaiian deep sea water. Here we compare BPR and GPR absorption spectra, photochemical reactions, and proton transport activity. The photochemical reaction cycle of Hawaiian deep ocean BPR in cells is 10-fold slower than that of GPR with very low accumulation of a deprotonated Schiff base intermediate in cells and exhibits mechanistic differences, some of which are due to its glutamine residue rather than leucine at position 105. In contrast to GPR and other characterized microbial rhodopsins, spectral titrations of BPR indicate that a second titratable group, in addition to the retinylidene Schiff base counterion Asp-97, modulates the absorption spectrum near neutral pH. Mutant analysis confirms that Asp-97 and Glu-108 are proton acceptor and proton donor, respectively, in retinylidene Schiff base proton transfer reactions during the BPR photocycle as previously shown for GPR, but BPR contains an alternative acceptor evident in its D97N mutant, possibly the same as the second titratable group modulating the absorption spectrum. BPR, similar to GPR, carries out outward light-driven proton transport in Escherichia coli vesicles but with a reduced translocation rate attributable to its slower photocycle. In energized E. coli cells at physiological pH, the net effect of BPR photocycling is to generate proton currents dominated by a triggered proton influx, rather than efflux as observed with GPR-containing cells. Reversal of the proton current with the K+-ionophore valinomycin supports that the influx is because of voltagegated channels in the E. coli cell membrane. These observations demonstrate diversity in photochemistry and mechanism among proteorhodopsins. Calculations of photon fluence rates at different ocean depths show that the difference in photocycle rates between GPR and BPR as well as their different absorption maxima may be explained as an adaptation to the different light intensities available in their respective marine environments. Finally, the results raise the possibility of regulatory (i.e. sensory) rather than energy harvesting functions of some members of the proteorhodopsin family. Microbial rhodopsins, seven-helix integral membrane proteins that use retinal as a chromophore, comprise a large family of photoactive proteins present in archaeal, bacterial, and eukaryotic microorganisms (1Béjà O. Spudich E.N. Spudich J.L. Leclerc M. DeLong E.F. Nature. 2001; 411: 786-789Crossref PubMed Scopus (649) Google Scholar, 2Spudich J.L. Yang C.S. Jung K.H. Spudich E.N. Annu. Rev. Cell Dev. Biol. 2000; 16: 365-392Crossref PubMed Scopus (507) Google Scholar, 3Sineshchekov O.A. Jung K.H. Spudich J.L. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 8689-8694Crossref PubMed Scopus (444) Google Scholar). Among the most abundant and widely distributed are the proteorhodopsins, the first of which was identified in the ubiquitous uncultured SAR86 group of marine proteobacteria in plankton from Monterey Bay coastal waters (4Béjà O. Aravind L. Koonin E.V. Suzuki M.T. Hadd A. Nguyen L.P. Jovanovich S.B. Gates C.M. Feldman R.A. Spudich J.L. Spudich E.N. DeLong E.F. Science. 2000; 289: 1902-1906Crossref PubMed Scopus (1159) Google Scholar). Nearly identical genes detected in picoplankton from the Southern Ocean (1Béjà O. Spudich E.N. Spudich J.L. Leclerc M. DeLong E.F. Nature. 2001; 411: 786-789Crossref PubMed Scopus (649) Google Scholar), Mediterranean, and Red Sea (5Man D. Wang W. Sabehi G. Aravind L. Post A.F. Massana R. Spudich E.N. Spudich J.L. Béjà O. EMBO J. 2003; 22: 1725-1731Crossref PubMed Scopus (245) Google Scholar) define a phylogenetic group of Monterey Bay-type pigment genes (clade I). The Monterey Bay pigment, a green light-absorbing proteorhodopsin (λmax 525 nm, hence referred to as “GPR” 1The abbreviations used are: GPR, green light-absorbing proteorhodopsin; BPR, blue-absorbing proteorhodopsin; MES, 4-morpholineethanesulfonic acid. (5Man D. Wang W. Sabehi G. Aravind L. Post A.F. Massana R. Spudich E.N. Spudich J.L. Béjà O. EMBO J. 2003; 22: 1725-1731Crossref PubMed Scopus (245) Google Scholar)), has been demonstrated to function as a light-driven proton pump (4Béjà O. Aravind L. Koonin E.V. Suzuki M.T. Hadd A. Nguyen L.P. Jovanovich S.B. Gates C.M. Feldman R.A. Spudich J.L. Spudich E.N. DeLong E.F. Science. 2000; 289: 1902-1906Crossref PubMed Scopus (1159) Google Scholar), and its transport, spectroscopic, and photochemical reactions have been characterized by a number of laboratories in Escherichia coli expressed forms (1Béjà O. Spudich E.N. Spudich J.L. Leclerc M. DeLong E.F. Nature. 2001; 411: 786-789Crossref PubMed Scopus (649) Google Scholar, 4Béjà O. Aravind L. Koonin E.V. Suzuki M.T. Hadd A. Nguyen L.P. Jovanovich S.B. Gates C.M. Feldman R.A. Spudich J.L. Spudich E.N. DeLong E.F. Science. 2000; 289: 1902-1906Crossref PubMed Scopus (1159) Google Scholar, 5Man D. Wang W. Sabehi G. Aravind L. Post A.F. Massana R. Spudich E.N. Spudich J.L. Béjà O. EMBO J. 2003; 22: 1725-1731Crossref PubMed Scopus (245) Google Scholar, 6Dioumaev A.K. Brown L.S. Shih J. Spudich E.N. Spudich J.L. Lanyi J.K. Biochemistry. 2002; 41: 5348-5358Crossref PubMed Scopus (188) Google Scholar, 7Dioumaev A.K. Wang J.M. Balint Z. Varo G. Lanyi J.K. Biochemistry. 2003; 42: 6582-6587Crossref PubMed Scopus (82) Google Scholar, 8Friedrich T. Geibel S. Kalmbach R. Chizhov I. Ataka K. Heberle J. Engelhard M. Bamberg E. J. Mol. Biol. 2002; 321: 821-838Crossref PubMed Scopus (200) Google Scholar, 9Krebs R.A. Alexiev U. Partha R. DeVita A. Braiman M.S. BMC Physiol. 2002; 2: 5Crossref PubMed Google Scholar, 10Lakatos M. Lanyi J.K. Szakacs J. Varo G. Biophys. J. 2003; 84: 3252-3256Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). In particular, its rapid photocycle, characteristic of transport rhodopsins, in E. coli membranes (4Béjà O. Aravind L. Koonin E.V. Suzuki M.T. Hadd A. Nguyen L.P. Jovanovich S.B. Gates C.M. Feldman R.A. Spudich J.L. Spudich E.N. DeLong E.F. Science. 2000; 289: 1902-1906Crossref PubMed Scopus (1159) Google Scholar) and in its native membranes from Monterey Bay plankton (1Béjà O. Spudich E.N. Spudich J.L. Leclerc M. DeLong E.F. Nature. 2001; 411: 786-789Crossref PubMed Scopus (649) Google Scholar) provides strong evidence that GPR functions as a light-driven proton pump photoenergizing SAR86 cells in their natural environment. A variant group (clade II) of proteorhodopsin genes differing by ∼22% in predicted primary structure from the clade I group has been detected in the Antarctic and deep ocean plankton from Hawaiian waters (1Béjà O. Spudich E.N. Spudich J.L. Leclerc M. DeLong E.F. Nature. 2001; 411: 786-789Crossref PubMed Scopus (649) Google Scholar). A member of clade II isolated from 75-meter deep plankton from the Hawaiian Ocean Time Station when expressed in E. coli exhibits a blue-shifted absorption spectrum (λmax 490 nm, hence referred to as “BPR”) with vibrational fine structure, unlike the unstructured spectrum of GPR (1Béjà O. Spudich E.N. Spudich J.L. Leclerc M. DeLong E.F. Nature. 2001; 411: 786-789Crossref PubMed Scopus (649) Google Scholar). A second clade II gene (from Antarctica) matches exactly these absorption properties when expressed in E. coli. 2W.-W. Wang, O. A. Sineshchekov, E. N. Spudich, and J. L. Spudich, unpublished data. Here we characterize the Hawaiian deep water BPR transport, spectroscopic, and photochemical reactions. We observe striking differences between the Hawaiian 75-meter BPR and the Monterey Bay GPR, a comparison with which provides insight into proteorhodopsin diversity in mechanism and possibly physiological function. Site-directed Mutagenesis—For simplicity, the Monterey Bay protein eBAC31A08 is designated GPR and Hawaiian Ocean Time Station variant HOT75m4 is designated BPR in this report. Mutants BPR_D97N and BPR_E108Q were constructed using two-step PCR (11Ho S.N. Hunt H.D. Horton R.M. Pullen J.K. Pease L.R. Gene (Amst.). 1989; 77: 51-59Crossref PubMed Scopus (6851) Google Scholar) following the procedure described for BPR_Q105L and GPR_L105Q (5Man D. Wang W. Sabehi G. Aravind L. Post A.F. Massana R. Spudich E.N. Spudich J.L. Béjà O. EMBO J. 2003; 22: 1725-1731Crossref PubMed Scopus (245) Google Scholar). All of the mutations were confirmed by DNA sequencing. Expression of Proteorhodopsins in E. coli Cells—GPR, BPR, and their mutants were expressed in E. coli strain UT5600 using plasmid pBAD-Topo as described previously (4Béjà O. Aravind L. Koonin E.V. Suzuki M.T. Hadd A. Nguyen L.P. Jovanovich S.B. Gates C.M. Feldman R.A. Spudich J.L. Spudich E.N. DeLong E.F. Science. 2000; 289: 1902-1906Crossref PubMed Scopus (1159) Google Scholar). Overnight cultures of transformants were diluted 1:50 and grown to 0.4 absorbance units at 600 nm at 30 °C. Cells were induced with 0.2% (+)-l-arabinose for3hat30 °C. To obtain protein for flash photolysis and proton transport measurements, all-trans-retinal was added to a final concentration of 5 μm with the arabinose. For measurement of retinal-generated absorption spectra, the apoproteins were induced without the addition of retinal. Preparation of Cell Membranes for Retinal-generated Spectra Measurements—Cells were disrupted by sonication, unlysed cells and large debris were removed by low speed centrifugation, and cell membranes were collected by ultracentrifugation at 180,000 × g for 1 h. The membranes were resuspended with 100 mm sodium acetate, MES, potassium phosphate (KPi), and Tris-HCl buffers at different pH values between 4.5 and 9, and retinal-generated absorption spectra were measured by adding all-trans-retinal from an ethanolic solution as described previously (5Man D. Wang W. Sabehi G. Aravind L. Post A.F. Massana R. Spudich E.N. Spudich J.L. Béjà O. EMBO J. 2003; 22: 1725-1731Crossref PubMed Scopus (245) Google Scholar, 12Bieszke J.A. Spudich E.N. Scott K.L. Borkovich K.A. Spudich J.L. Biochemistry. 1999; 38: 14138-14145Crossref PubMed Scopus (139) Google Scholar). Proton Pumping Measurements—Right-side-out membrane vesicles were prepared as follows. 100 ml of induced cells were harvested by centrifugation at 3600 × g for 10 min, resuspended into 10 ml of 30 mm Tris-HCl, pH 8.0, and 20% sucrose. 100 μg (104 units) of lysozyme was added and stirred gently at room temperature. Spheroplasts were collected by centrifugation at 3600 × g for 15 min at room temperature and resuspended in 1 ml of buffer (100 mm KPi, pH 7.0, 20 mm MgSO4, 20% sucrose, and 4 mg of DNase) and injected using a 1-ml syringe (18-gauge needle) into 500 ml of a rapidly stirring solution of 50 mm KPi, pH 7.0, at 37 °C. After 15 min of gently stirring at 37 °C, Na-EDTA was added to a final concentration of 10 mm and stirred for another 15 min, after which MgSO4 was added to a final concentration of 15 mm, followed by 15 min of stirring. Cell debris was removed at 3600 × g for 10 min, and vesicles were collected at 16,000 × g for 1 h and resuspended in 1 ml of 100 mm KPi, pH 7.0, and 10 mm MgSO4. For the measurement of proton pumping of cells, the E. coli cells with expressed proteorhodopsins were pelleted at 3600 × g for 10 min at room temperature and with solution mm 10 mm 100 μm at room temperature. were with 100 at 500 20 nm using a in with and and The pH was with a pH absorption were measured a flash photolysis to that used in 12Bieszke J.A. Spudich E.N. Scott K.L. Borkovich K.A. Spudich J.L. Biochemistry. 1999; 38: 14138-14145Crossref PubMed Scopus (139) Google Scholar). The flash was from a nm, from the were by a at 20 and in a using the from with a flash were To of with the were and from the The was used for For flash membranes were prepared as described and in 50 mm Tris-HCl buffer at pH and E. coli cells with expressed proteorhodopsins were pelleted at 3600 × g for 10 min at room temperature and and with 50 mm Tris-HCl at pH and of the and absorption spectra of proteorhodopsin apoproteins in E. coli membrane were measured by difference as described previously (5Man D. Wang W. Sabehi G. Aravind L. Post A.F. Massana R. Spudich E.N. Spudich J.L. Béjà O. EMBO J. 2003; 22: 1725-1731Crossref PubMed Scopus (245) Google Scholar, 12Bieszke J.A. Spudich E.N. Scott K.L. Borkovich K.A. Spudich J.L. Biochemistry. 1999; 38: 14138-14145Crossref PubMed Scopus (139) Google Scholar). After adding the retinal the spectra were with an 10 min for after which absorption The spectra were used for the of the as follows. The at different pH values were measured to the spectra of the membrane with proteorhodopsin apoproteins The position of the absorption maxima of the of and forms at different pH values the of the forms because of different and of their For the of the a which the concentration of and forms with the position of the absorption of their was by of absorption spectra of and forms with The of and forms at different pH values was using this from the position of absorption of their and with functions BPR of the most properties of retinal as a is the large of its absorption spectrum with the Biophys. PubMed Scopus Google Scholar, M. M. J. Biol. 1989; PubMed Scopus Google Scholar). In rhodopsins, retinal is to the group of a a retinylidene Schiff In a retinylidene Schiff base exhibits a of the protein the Spudich J.L. S. K. J. Biol. Full Text Full Text PDF PubMed Scopus Google to to 490 nm in BPR and 525 nm in Microbial in spectral forms in a a blue-shifted and The in absorption by nm the pigment and of In S. J. Biol. Full Text PDF PubMed Google Scholar), I Spudich J.L. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar) and II J. Spudich E.N. M. Spudich J.L. PubMed Scopus Google Scholar), and GPR A.K. Brown L.S. Shih J. Spudich E.N. Spudich J.L. Lanyi J.K. Biochemistry. 2002; 41: 5348-5358Crossref PubMed Scopus (188) Google Scholar), the spectral is attributable to the of the primary retinylidene Schiff base counterion in Asp-97 in because this residue to the in of these the and the absorption spectrum in the We BPR in E. coli membranes to its and spectra and the of the spectral a of nm a than the 20 nm in GPR (5Man D. Wang W. Sabehi G. Aravind L. Post A.F. Massana R. Spudich E.N. Spudich J.L. Béjà O. EMBO J. 2003; 22: 1725-1731Crossref PubMed Scopus (245) Google Scholar) and a of vibrational fine structure in the spectrum as observed for II T. K. Spudich J.L. Biochemistry. PubMed Scopus Google Scholar). The GPR spectral well to a of a second is evident in the BPR to to the that a with values and unlike other microbial the of the Schiff base counterion Asp-97 to an but rather a with a of in the BPR_D97N among microbial that an titratable group modulates the of the in of the concentration of forms of GPR BPR and BPR_D97N The of were from the of absorption maxima using a and and values were by of the with The pH in BPR be with as a in values are shown in the The of the in are that the of BPR from the spectral for R. S. Biochemistry. PubMed Scopus Google Scholar) and I Spudich J.L. Biochemistry. 2002; 41: PubMed Scopus Google Scholar). The in are attributable to the of the of the retinylidene Schiff base counterion in and in which to Asp-97 in BPR) the of another residue in the In of the counterion residue with the spectral and the evident in BPR_D97N BPR an of than in flash photolysis of the BPR pigment in E. coli cells differences in of the BPR photocycle from that of GPR In cells, absorption difference spectra in the of nm show the and of the by the and of an intermediate at of nm and very at nm a deprotonated intermediate is to The of absorption by the of the is slower than that for GPR in cells the same In contrast to GPR, the blue-shifted intermediate in BPR in very low with of of the of the low of accumulation and the very nm in cell the of is nm by the of the In we detected the and of BPR at nm, its is to because of by a of absorption in the of was observed in BPR in and in membranes of the BPR_E108Q Asp-97 and Glu-108 as a Proton and for Schiff Proton during the BPR of absorption near nm is of an retinylidene Schiff base in this BPR In GPR, Asp-97 is the proton acceptor from the Schiff base in the photocycle and in is A.K. Brown L.S. Shih J. Spudich E.N. Spudich J.L. Lanyi J.K. Biochemistry. 2002; 41: 5348-5358Crossref PubMed Scopus (188) Google Scholar). BPR_D97N a reduced accumulation of a similar of Asp-97 as a proton acceptor in BPR some is evident in that an alternative proton acceptor residue is present in this the titratable group that the absorption of BPR_D97N is the alternative proton In GPR, Glu-108 is the proton to the Schiff base for the proton transfer during the of the intermediate to as by a in The photocycle in BPR is in a similar as a Schiff base proton for this residue in the BPR photocycle. most accumulation is in the without accumulation of and In the was the intermediate is with accumulation of the accumulation of the absorption of the of which is nm than that of the intermediate in The at the of in the predicted of we have that a residue in GPR in BPR) the absorption spectra of the GPR_L105Q is blue-shifted and vibrational fine structure, BPR_Q105L is to green absorption and fine structure in its spectrum (5Man D. Wang W. Sabehi G. Aravind L. Post A.F. Massana R. Spudich E.N. Spudich J.L. Béjà O. EMBO J. 2003; 22: 1725-1731Crossref PubMed Scopus (245) Google Scholar). In addition to as a spectral the of residue between GPR and BPR has the of the the photocycle of GPR is than that of BPR with evident accumulation unlike BPR when at nm The in GPR the photocycle to a of BPR with at The in BPR the photocycle but accumulation of in the the residue at position as a for spectral and photochemical properties of GPR and BPR, but differences in addition to that at position to the rapid photocycle of Proton in E. coli and has shown that GPR exhibits of from E. coli cells (4Béjà O. Aravind L. Koonin E.V. Suzuki M.T. Hadd A. Nguyen L.P. Jovanovich S.B. Gates C.M. Feldman R.A. Spudich J.L. Spudich E.N. DeLong E.F. Science. 2000; 289: 1902-1906Crossref PubMed Scopus (1159) Google Scholar), and evidence that GPR functions as a light-driven proton pump (1Béjà O. Spudich E.N. Spudich J.L. Leclerc M. DeLong E.F. Nature. 2001; 411: 786-789Crossref PubMed Scopus (649) Google Scholar, 4Béjà O. Aravind L. Koonin E.V. Suzuki M.T. Hadd A. Nguyen L.P. Jovanovich S.B. Gates C.M. Feldman R.A. Spudich J.L. Spudich E.N. DeLong E.F. Science. 2000; 289: 1902-1906Crossref PubMed Scopus (1159) Google Scholar, 5Man D. Wang W. Sabehi G. Aravind L. Post A.F. Massana R. Spudich E.N. Spudich J.L. Béjà O. EMBO J. 2003; 22: 1725-1731Crossref PubMed Scopus (245) Google Scholar, 6Dioumaev A.K. Brown L.S. Shih J. Spudich E.N. Spudich J.L. Lanyi J.K. Biochemistry. 2002; 41: 5348-5358Crossref PubMed Scopus (188) Google Scholar, 8Friedrich T. Geibel S. Kalmbach R. Chizhov I. Ataka K. Heberle J. Engelhard M. Bamberg E. J. Mol. Biol. 2002; 321: 821-838Crossref PubMed Scopus (200) Google Scholar, 9Krebs R.A. Alexiev U. Partha R. DeVita A. Braiman M.S. BMC Physiol. 2002; 2: 5Crossref PubMed Google Scholar) in its natural marine (1Béjà O. Spudich E.N. Spudich J.L. Leclerc M. DeLong E.F. Nature. 2001; 411: 786-789Crossref PubMed Scopus (649) Google Scholar). Here we the transport of GPR and BPR in E. coli vesicles and cells. In E. coli and BPR outward proton transport similar pigment of GPR and BPR, are at a slower rate in BPR, as from its slower photocycle In with a of GPR proton transport A.K. Wang J.M. Balint Z. Varo G. Lanyi J.K. Biochemistry. 2003; 42: 6582-6587Crossref PubMed Scopus (82) Google Scholar), we observe the of pumping by pH in vesicles in cells, to has been for GPR in a T. Geibel S. Kalmbach R. Chizhov I. Ataka K. Heberle J. Engelhard M. Bamberg E. J. Mol. Biol. 2002; 321: 821-838Crossref PubMed Scopus (200) Google Scholar). pH cells with GPR and BPR out light-driven proton at pH is a net proton influx for the cells with BPR GPR proton but an net proton influx is observed following E. coli cells without proteorhodopsin proton is that the pumping by BPR the of proton channels in the E. coli which results in proton influx by the and of the membranes of the energized E. coli cells. A similar was for proton in membranes to of an and W. Biophys. PubMed Scopus Google Scholar). We proton in E. coli cells with valinomycin and which the of the by the transport by the of proton induced by BPR at pH is (i.e. to proton by of of the proton by the confirms that the proton are currents to transport of another by BPR We the different proton from BPR and GPR photocycling as the of the slower rate of proton of In cells, the proton currents are dominated by the triggered proton influx in GPR-containing cells, the triggered influx is by the proton rate of the this low light of GPR, which the rate of proton to that of BPR at results in a proton influx similar to that of BPR in of GPR and BPR difference in photocycle rates between GPR and BPR and their different absorption maxima may be explained as an adaptation to the different light intensities in their respective marine measured spectral of intensities of at the ocean and at depths Scholar). of at 525 nm absorption of the ocean using a of GPR and we that 1 be available for absorption for this a of GPR and the GPR photocycle rate is well to the fluence the at which the BPR gene was the at this is and the available for absorption the absorption spectrum are due to the very in light at 500 of the of to the the spectral to nm in BPR this in available light energy but at most 1 be available for this photon fluence rate a 10-fold slower photocycle in BPR than in GPR as we observe is for a photocycle than that of BPR at that BPR may function to cells by light-driven proton pumping as we a of energy from BPR, which is by the low light intensities in deep In E. at neutral pH the of observed in GPR and BPR, be by proton from the cell at light intensities because of its may indicate a regulatory rather than function of BPR, which may be of proton channels in to the in membrane and of A function be of cell light due to is the cells BPR, we that BPR may be by as are with a present in the E. coli membrane. In this BPR may its light-driven proton transport in its transport is by as is the with the R.A. W. I. E. Spudich J.L. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, G. Engelhard M. G. Bamberg E. Proc. Natl. Acad. Sci. U. S. A. 2001; PubMed Scopus Google Scholar, M. K. M. N. Biophys. J. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar).