- Email: [email protected]
Measurement of proton release and uptake by analogs of bacteriorhodopsin
Bioelectrochemistry 51 Ž2000. 27–33 www.elsevier.comrlocaterbioelechem
Measurement of proton release and uptake by analogs of bacteriorhodopsin Howard H. Weetall a,) , Anna Druzhko a , Angel R. de Lera b,1, Rosana Alvarez b,1, Baldwin Robertson a a b
Biotechnology DiÕision, National Institute of Standards and Technology, Gaithersburg, MD 20899, USA Department of Organic Chemistry, UniÕersity of Santiago de Compostela, Santiago de Compostela, Spain Received 25 March 1999; received in revised form 14 October 1999; accepted 17 October 1999
Abstract Proton release and subsequent uptake by several forms of bacteriorhodopsin ŽbR., including 4-keto analogs of wild-type ŽWT. and D96N and D85N mutants as well as the 9-demethylretinal analog of WT and D96N mutants, have been measured using a highly sensitive electrochemical technique. Release and uptake of protons by bR in membrane patches on a tin oxide electrode produce a current transient whose amplitude is proportional to the rate of pH change at the electrode surface. Profiles of proton release by the analogs vs. pH are substantially different from the profiles of the native proteins. q 2000 Elsevier Science S.A. All rights reserved. Keywords: Proton; Analog; Bacteriorhodopsin
1. Introduction Bacteriorhodopsin ŽbR. is a protein–chromophore complex that, when it absorbs yellow light, releases protons on the periplasmic side of the membrane and subsequently takes them up from the cytoplasmic side, thus pumping them across the membrane w1x. Retinal, the chromophore that is bound to Lys-216 of the protein by a protonated Schiff base, is responsible for the absorption that occurs at a maximum of approximately 570 nm. A quantum of yellow light induces a trans–cis isomerization of the retinal, followed by transitions of the protein–retinal complex through a number of intermediate states ŽI, J, K, L, M, N, and O. that are characterized by different protonation states of the Schiff base and of several amino acid residues within the protein w2,3x. The M state of the photocycle is formed when, as a result of the isomerization of retinal, the proton on the Schiff base is transferred to Asp-85, and subsequently a proton from another amino
)
Corresponding author. Tel.: q1-301-975-2000; fax: q1-301-5481087. E-mail address: [email protected] ŽH.H. Weetall.. 1 Present address: Universidad de Vigo, Departmento de Quimica Pura y Aplicada, Facultad de Ciencias, As Lagsas Marcosende, Vigo 36200, Spain.
acid XyŽ possibly Arg-82. w4x is released into the solution on the periplasmic side of the membrane and Asp-96, in turn, is reprotonated from the cytoplasmic solution. The proton release generally occurs within 50 ms after absorption of a quantum of light energy. The proton uptake in wild-type ŽWT. bR requires 10–20 ms. In D96N, the M state decays only when the Schiff base is reprotonated from solution, which can take over 100 s depending on pH w5–7x. In the next step, for both WT and D96N bR, retinal reisomerizes back to the all-trans configuration, and finally the proton from Asp-85 reprotonates the proton release group Xy, returning the complex to its initial state w2,3x. The D85N mutant, lacking the Asp in the 85 position, cannot accept a proton, so any release of a proton from the Schiff base must be by diffusion. This mutant shows the most dramatic pH dependence, because the pK of its Schiff base protonationrdeprotonation is approximately 6–8 w8,9x, compared with 13 observed for the WT w10–12x. Very little is known of the proton release function in this mutant. A study on purple membranes ŽPms. of D85N attached to a lipid bilayer w9x showed that a photocurrent can be generated in yellow light. The mechanism appears similar to that observed with halorhodopsin. Halorhodopsin can act as an inwardly directed proton pump after sequential absorption of a yellow and a blue photon w13x. This is due to a side reaction of the photocycle. At neutral pH, the
0302-4598r00r$ - see front matter q 2000 Elsevier Science S.A. All rights reserved. PII: S 0 3 0 2 - 4 5 9 8 Ž 9 9 . 0 0 0 7 2 - 0
28
H.H. Weetall et al.r Bioelectrochemistry 51 (2000) 27–33
Schiff base can deprotonate after absorption of a yellow photon, forming an M-like state. The proton is released on the cytoplasmic side. Blue light reprotonates the Schiff base from the extracellular side, completing the proton translocation. This translocation is amplified by azide, which increases the formation of the M-like state upon illumination with yellow light. For D85N, the Schiff base has a pK of around 8. In the dark at physiological pH, a portion of D85N has a deprotonated Schiff base with retinal in the all-trans configuration and a portion has it in the 13-cis configuration w14x. Absorption of a yellow photon and then a blue one produces inwardly directed proton transfer similar to that observed for halorhodopsin. The proton transfer of D85N, like that of halorhodopsin, is also enhanced by the addition of azide. Studies in this laboratory on D85N unoriented films indicate that at neutral pH, proton release occurs after exposure to blue light followed by yellow light. The measurement of proton release vs. pH by modified and unmodified forms of D96N and D85N can give important information on the mechanism of proton release by bR. Much effort has been made to characterize the 4-keto derivative of bR w13,15–19x. The transient electrical current that occurs when a photon is absorbed by 4-keto bR in air has been measured w20x. However, the specific charges whose displacement generates the current have not been identified. No measurement of the translocation of protons, as distinguished from other electrical charges, has been carried out. The intermediates of the photocycle of 4-keto WT bR have so far been identified only by their optical absorption spectra w15,18x. Even less is known about the 4-keto derivative of D85N. The mutant form of 9-demethylretinal has not been examined for photocycle activity or proton release. For 4-keto D96N and WT derivatives, the major optical characteristics are similar to those observed in gelatin films w19x. An initial absorbance peak at approximately 508 nm decays on exposure to yellow light and peaks appear at 390, 420 and 440 nm. These three peaks have been labeled as corresponding to M-like states w19x. Formation of the three peaks can occur in 60 ms for the 4-keto-WT, while in the 4-keto D96N, mutant in suspension requires several milliseconds w18x; they both require several milliseconds when the bR derivatives are in membrane patches in dried film w19x. Thus, release of protons by this derivative and the effect of pH on the release are of great interest. Since the characterization of retinal derivatives can be an important tool in understanding retinal–protein interactions, we have also chosen to examine the 9-demethylretinal derivatives of WT and D96N bR. The 9-demethyl analog has been described as binding to the apoprotein of bR with only minor structural changes w21x. Unlike the 4-keto analog, this analog has a modification along the backbone of the retinal chain rather than on one of the rings, where one would expect a greater effect. Informa-
tion related to proton release and uptake of this bR derivative will be helpful in determining the mechanism of action of both the 9-demethyl and the 4-keto derivatives.
2. Materials and methods Pms from the mutant and WT strains of Halobacterium salinarium were prepared by standard protocol w21x. An aqueous suspension of WT and D96N was illuminated by light at 500 nm in the presence of 0.5 M NH 3 OH, at pH 8.2–8.4 at 78C. In the case of the D85N mutant, which has a 20-fold lower reaction rate with NH 2 OH w22x, the apomembranes of this mutant were prepared differently. The suspension of the D85N mutant was illuminated by yellow light in the presence of 1 M NH 2 OH, 100 mM NaCl, and 25 mM Trizma base buffer, pH 8.6 at 228C. The activity of the apomembranes was determined by reconstruction with all-trans retinal ŽSigma, St. Louis, MO.. The 4-ketoretinal ŽFig. 1. was supplied by A.A. Khodonov ŽM.V. Lomonosov Institute of Fine Chemical Technology, Moscow, Russia.. For reconstruction, the analogs were first dissolved in isopropanol before adding to the apoprotein membranes in aqueous suspension. The 9-demethylretinal derivative ŽFig. 1. was prepared as previously described w23x. In several experiments, the retinal oxime was removed by extraction with bovine serum albumin as described by Katre et al. w23x before or after adding the retinal analog. This was carried out to determine the effect of retinal oxime on proton release and uptake. A film of membrane patches was prepared by depositing 10 ml of an aqueous suspension of membrane patches containing bR on an antimony-doped tin oxide-coated glass slide by placing the drop on the slide and letting the solution dry to form a spot approximately 6 mm " 1 mm in diameter. The concentration of the bR derivatives used for the pH studies varied from 0.1 to 1.0 mgrL.
Fig. 1. Structural formulas for the 9-demethylretinal Žtop. and the 4-ketoretinal Žbottom..
H.H. Weetall et al.r Bioelectrochemistry 51 (2000) 27–33
The tin oxide was used as the working electrode in an electrochemical cell ŽBAS CC-4 Flow Cell. modified to hold a working electrode 0.5 = 1.5 in.2 in size. A Lucite cover compressed the glass electrode and a Teflon gasket against the metal body of the cell, the latter serving as the counterelectrode. The Lucite cover was later replaced with a black nylon cover having a hole 5 mm in diameter directly over the 6 mm bR spot. The active area of the bR film was the spot on the tin oxide that was illuminated through the Lucite cover, or for later experiments, through the 5 mm hole in the black nylon cover. Using the opaque cover with the hole made the area more reproducible. The Teflon gasket was 0.25 mm thick and had a slot Žwith semicircular ends. cut in it that was 17 mm long and 4.5 mm wide. Thus, the walls of the cell were: tin oxide on the front where the light entered, the metal counterelectrode on the rear, and the Teflon on the bottom, top, and sides. By calculation, the volume of the flowcell was approximately 18 ml so that five drops washed the cell 15 times. Access to the reference electrode was through an approximately 0.2-mm hole in an insulating plug in the metal counterelectrode. The electrolyte was a mixture of three buffers: potassium citrate, potassium phosphate, and potassium borate, each 0.005 M in concentration plus 0.05 M potassium chloride. The pH of the solutions was adjusted with hydrochloric acid or potassium hydroxide. The reference electrode was silverrsilver chloride ŽBAS MW 2021.. The cell was connected to a potentiostat consisting of an operational amplifier ŽOPA111. with a 1 M V resistor in a feedback circuit. The voltage across the resistor was amplified by an instrumentation amplifier ŽAD524. with gain s 1. The amplifiers were battery-powered and completely enclosed along with the electrochemical cell in solid aluminum shielding to reduce electrical interference. The output was connected by coaxial cable to an oscilloscope ŽTektronix TDS 410., that could be set from 1.00 to 0.5 mVrbox, and 500 data points were collected in 2 s, so the response time for the system of approximately 4 ms. A horizontal beam of light from a mercury-arc lamp ŽLEP, HBO 100. was filtered by a 475-nm long pass filter ŽOriel 59490. and focused through a hole in the shielding onto the tin oxide electrode. The rest of the apparatus used were similar to that described previously w24x.
3. Results and discussion Under intense yellow light, retinal in the presence of excess hydroxylamine is photolyzed and reacts to form retinal oxime. Pms treated in this manner lose their color. When the chromophore analog is added, reconstitution of the bR molecule occurs, with the new chromophore attached to the protein and the retinal oxime still remaining but unattached. Since there is the possibility that in time an
29
exchange might occur between the natural retinal oxime and the analog bound to the opsin, reconverting the bR back to its natural state, any proton release or uptake observed might be attributable to reversion product. However, when the retinal oxime is removed from the opsin before introduction of the chromophore analog, there is no chance for reconversion to occur, and one can be confident that no natural chromophore bR is present. Measurement of the proton release and uptake of WT bR opsin after removal of the retinal oxime showed responses identical to that observed before removal of the unbound oxime. This response did not exceed 1 nA, which is about as small a current as we can observe. Thus, all other experiments were carried out without removing the retinal oxime, since its presence evidently had no effect. The proton release and uptake functions were measured by observing the amplitude of a light-induced current transient generated by the potentiostat connected to the electrochemical cell. The current rises because bR in the Pm patches on the tin oxide electrode releases protons near the electrode surface w24x. There are two physical effects that can result from motion of ions or charged complexes that occur when light is absorbed by bR. The first is a charge-displacement current and the second is a transient current due to the pH change that results from the release and uptake of protons. The voltage generated by bR on a tin oxide electrode in air is of the first kind; it is due to charge displacement and leads to an observed current w17x. However, this current is too small to be detected in our system. The current generated by the potentiostat when light excites the bR is of the second kind. It is proportional to the rate of change of the pH produced at the electrode surface by the protons released or taken up w23x. This provides a highly sensitive measurement of transient pH changes much more sensitive than can be achieved with a typical pH meter. However, the most important distinction between the two mechanisms is that measurement of the charge-displacement current does not reveal anything about just which ions or charged complexes are the ones that move and produce the current. Any charge that moves produces current. In contrast, the current observed using the tin oxide pH-sensitive electrode in the electrochemical cell is due specifically to release or uptake of protons. Both the proton-release transient that occurs when light is turned on and the proton-uptake transient that occurs when the light is turned off provide a direct measurement of stages of the proton-transport function of bR. Since the studies were carried out with randomly oriented patches, approximately half of the Pms has the cytoplasmic side toward the electrode and half has the periplasmic side toward the electrode. When the light is turned on, both release and uptake of protons occur near the electrode surface. However, there is a delay between the proton release and the subsequent uptake. Deprotonation requires 50 ms while reprotonation requires approximately 10 ms so the current due to proton release over-
30
H.H. Weetall et al.r Bioelectrochemistry 51 (2000) 27–33
whelms the current due to the slower uptake. When the light is turned off, there is only proton uptake and the current is in the opposite direction. We measured the amplitudes of photoinduced transient current generated by the 4-keto analogs of WT, D96N, and D85N. We chose to examine these because the mutations have profound effects when the chromophore is natural retinal. Asp-96 is the donor in the reprotonation of the Schiff base and Asp-85 is the acceptor in its deprotonation. We included the 9-demethylretinal because one might expect little change in the properties of this analog as compared to the 4-keto analog. This gives us some information on the effects attributable to changes in different parts of the chromophore since the 9-demethylretinal is modified on the polyene side chain and the 4-ketoretinal is modified on the ring structure ŽFig. 1.. We have chosen to examine proton release over a pH range of 3–9 because it is well-known that the pK of the Schiff base of several of the retinal analogs is smaller than for the natural chromophore for which the pK of the Schiff base is between 12 and 13 w10–12x. In the case of most of these analogs, the pK of the Schiff base evidently has been decreased to between 6 and 9 w10–12x. Studying pH profiles of both natural retinal bR and analog bR should give us greater insight into the mechanism of the proton release and uptake. The pH profiles of the 4-ketoretinal-WT, 4-ketoretinalD96N, 9-demethylretinal-WT, and the 9-demethylretinalD96N derivatives are shown in Fig. 2. Each of these derivatives shows a reversal in the proton release and uptake current transient at acid pH. This suggests that, as previously reported w10–12x, the pK of the Schiff base in the analogs must be lower. For the 9-demethylretinal derivative, deprotonation of the Schiff base has been eliminated or slowed to such a rate that the uptake completely dominates the release. It has also been suggested that the pK at the 85 position in similar analogs changes from 3.2 to above 9 w11x. Thus, the 85 position tends to remain protonated. For the 4-keto D85N, we could not observe any proton transfer under the conditions we examined. This may be because no more then 10% of the apoprotein was reconstituted with the 4-ketoretinal. The native form of D85N shows an M-like spectral peak in the dark because a fraction of the chromophore is in the all-trans configuration. Blue light causes decay of the M-like state and the return of the chromophore to the cis configuration. Turning on yellow light causes proton release as indicated by the appearance of a transient photocurrent whose direction corresponds to a decrease of pH at the electrode surface. Removal of the light causes an immediate proton uptake away from the electrode surface. Since the film is unoriented, the experiment does not show whether the uptake occurs on the cytoplasmic side and the release occurs on the extracellular side of the Pm. However, Tittor et al. w9x, using bilipid-oriented membrane vesicles, observed a transient photocurrent upon excitation
Fig. 2. The normalized pH profiles of the light-on transient current peaks for WT and mutant bR analog. Positive currents represent net proton release, negative currents represent net proton uptake. The current due to proton release was normalized by dividing by the current at the optimum pH for each derivative. The relative current at optimum pH for each analog was taken as 1.00. Open triangles represent 9-demethylretinal WT, closed circles represent 4-ketoretinal D96N, and open circles represent 4-ketoretinal WT. The inverted triangles represent 9-demethylretinal D96N. The buffer used in all studies was 0.005 M each of potassium citrate, potassium phosphate, potassium borate and 0.05 M potassium chloride with pH adjusted by adding hydrochloric acid or potassium hydroxide solutions.
with yellow light. They believe that the transient current was inwardly directed. This was determined on the assumption that the WT Pms and the Pms of the mutants absorbed preferentially onto the planar bilayer with the extracellular side toward the bilayer. The oriented lipid bilayers and the unoriented films appear to show similar results with yellow light. In both cases, one observes a transient photocurrent in the same direction, indicating proton release, followed by a second transient response in the opposite direction on removal of the light source. The D85N Pms in the lipid bilayer release protons on the cytoplasmic side w9x. In the unoriented films, we see proton release toward the electrode surface which is consistent with release of protons on the cytoplasmic side, since half of the patches is oriented with the cytoplasmic side toward the electrode. The side of the release will be confirmed in a planned study of oriented films. The natural-chromophore D85N bR photocurrent transient was examined as a function of pH. The pK of the Schiff base in D85N is approximately 8 w8,9x. The pH increase from 3 to 9 significantly shifts the equilibrium of the ground state and the M-like state towards the M-like
H.H. Weetall et al.r Bioelectrochemistry 51 (2000) 27–33
Fig. 3. The normalized pH profiles of light-on transient current peaks for bRs containing natural chromophore Žretinal.. Positive currents represent net proton release, negative currents represent net proton uptake. The current due to proton release was normalized by dividing the current at the optimum pH for each derivative. The relative current at optimum pH for each bR preparation was taken as 1.00. The X represents the WT, the plus represents D85N, and the closed triangle represents D96N. The buffer used in all studies was 0.005 M potassium citrate, potassium phosphate, potassium borate and 0.05 M potassium chloride with pH adjusted by adding hydrochloric acid or potassium hydroxide solutions.
state at higher pH. The normal rise of the M-like state is known to be blocked in D85N w8x. However, minor amounts of the intermediate are formed due to other mechanisms, including direct deprotonation to the medium. Alternatively, for pH close to the pK of the Schiff base, light
31
excitation may lower the pK and shift the equilibrium of the Schiff base towards deprotonation w8x. The presence of a photoinduced current at a pH between 5 and 8 suggests that this may be the case. The pH profiles of the 4-ketoretinal and the 9-demethylretinal derivatives of the D96N look very similar ŽFig. 2.. The differences in the retinal substitution appear to play less of a role in determining the pH characteristics of these derivatives than does the amino acid substitution. When comparing the natural-chromophore D96N with the two analogs, little difference is observed ŽFig. 2.. If one examines the pH transient current profiles for bRs containing the native chromophore ŽFig. 3., one observes smaller reversals of the current transient for both WT and D96N at acid pH ŽFig. 3.. As previously mentioned for D96N, there is a smaller difference between the pH profiles of the 4-ketoretinal analog and the 9-demethylretinal analog and the native form. This suggests that the absence of Asp-96 plays a far more important role in determining the proton uptake and release mechanism then does the chromophore. The 9-demethylretinal analogs were added to this study because the removal of the methyl group on the polyene portion of the retinal should have a small effect, compared to the 4-ketoretinal analog, where substitution is made on the ring structure. The pH profiles of the analogs of the WT derivatives showed greater reversal of the light-induced current transient for pH lower than observed for the D96N analogs. This suggests that in the WT, the chromophore plays a larger role in determining the characteristics of proton release and uptake than in the D96N analogs. Computational studies w21x suggest that the location of the methyl groups on the polyene side chain determines the overall shape of the retinal ligand. According to the suggested model w14x, the effect of the methyl group should be purely steric in nature. It is suggested that Trp-182 contacts the C9-methyl group, and that this contact is critical for the photocycle function. Our data suggest that the effects of the removal of the 9-methyl functional-
Table 1 Sample
pH optimum
Peak current a mean ŽnA.
Peak current standard deviation ŽnA.
WT WT 4-keto WT 9-demethyl D96N D96N 4-keto D96N 9-demethyl D85N D85N 4-keto
7.0 6.5 6.5 6.5 6.5 6.0 7.0 Not detectable
220 23 116 400 29 74 18 Not detectable
12 2 6 18 2 5 3 Not detectable
a The values shown here represent the light on current density transient only. The peak currents were determined at the optimum pH for proton release. Samples were irradiated with blue light for 1 min before a 1-s flash of yellow light. All samples contained 0.5 mgrml of bR in concentration. Sample size was 20 ml. The sample labeled NA did not show detectable proton release or uptake. The standard deviations of the peak current values are for five replicates on the same electrode. Each sample film was characterized on separate electrodes. The uncertainty for bR on different electrodes is 20% of the peak currents.
32
H.H. Weetall et al.r Bioelectrochemistry 51 (2000) 27–33
ity may have a larger effect on the protein than computational studies anticipate. We have also compared the peak values for the proton release current transients for each of the bRs at equal protein concentrations ŽTable 1.. The results show that the greatest magnitude was observed with D96N while the smallest value was observed with D85N. These data are contrary to that of Le Coutre et al. w25x. These studies using D pH measurements in bR liposomes showed that D96N had ) 3% of the proton pumping activity of the WT. However, the peak current for proton release depends on the turnover rate and since the total turnover time of the WT photocyle is short in comparison with the time scale of the current transient. Since proton uptake begins earlier when the turnover is rapid, this diminishes the net magnitude of the observed current transient peak. In the case of the slower mutants and analogs, the peak current transients are more closely related to the ground to M-state transition efficiency because the M-to-ground state transition is much slower than the time scale of the peak current transient. As previously stated, the current transient is directly related to the rate of proton transport. We have examined and compared the proton uptake and release of WT and two mutant bRs containing both native and artificial chromophores. Substitution of either 4-ketoretinal or 9-demethylretinal for the native chromophore substantially reduces the proton transport by WT at acid pH, but only slightly reduces the proton release by D96N.
w5x
w6x
w7x
w8x
w9x
w10x
w11x
w12x
w13x
w14x
Acknowledgements w15x
This work was supported by the National Institute of Standards and Technology. The work also received financial support from FIS Žcontract 95r1534., the Xunta de Galicia Žgrant XUGA20904B95., and the University of Santiago de Compostela Žfellowship to R. Alvarez.. The authors wish to thank Dr. Tonya Herne from the National Institute of Standards and Technology for her valuable suggestions while reviewing this manuscript.
References w1x D. Oesterhelt, W. Stockenius, Rhodopsin-like protein from the purple membrane of Halobacterium halobium, Nat. New Biol. 233 Ž1971. 149. w2x M. Kono, S. Misra, T.G. Ebrey, pH dependence of light-induced proton release by bacteriorhodopsin, in: M.B. Jackson ŽEd.., Thermodynamics of Membrane Receptors and Channels, CRC Press, Cleveland, 1993, p. 353. w3x R.A. Mathies, S.W. Lin, J.B. Ames, W.T. Pollard, From femtoseconds to biology: mechanism of bacteriorhodopsin’s light-driven proton pump, Annu. Rev. Biophys. Biophys. Chem. 20 Ž1991. 491. w4x S.P. Balashov, R. Govindjee, M. Kono, E. Imasheva, E. Lukashev, T.G. Ebrey, R.K. Crouch, D.R. Menick, Y. Feng, Effect of the arginine-82 to alanine mutation in bacteriorhodopsin on dark adapta-
w16x
w17x
w18x
w19x
w20x
w21x
w22x
tion proton release and the photochemical cycle, Biochemistry 32 Ž1993. 10331. A. Miller, D. Oesterhelt, A guide to electron paramagnetic resonance spectroscopy of photosystem II membranes, Biochim. Biophys. Acta 1020 Ž1990. 57. M. Holz, L.A. Drachev, T. Mogi, H. Otto, A.D. Kaulen, M.P. Heyn, V.P. Skulachev, H.G. Khorana, Replacement of aspartic acid-96 by asparagine in bacteriorhodopsin slows both the decay of the M intermediate and the associated proton movement, Proc. Natl. Acad. Sci. U.S.A. 86 Ž1989. 2167. J. Tittor, C. Soell, D. Oesterhelt, H.-J. Butt, E. Bamberg, A defective proton pump, point-mutated bacteriorhodopsin Asp-96 . . . Asn is fully reactivated by azide, EMBO J. 8 Ž1989. 3477. H. Otto, T. Marti, M. Holz, T. Mogi, L.J. Stern, F. Engel, H.G. Khorana, M.P. Heyn, Substitution of amino acid Asp-85, Asp-212, and Arg-82 in bacteriorhodopsin affects the proton release of the pump and the pK of the Schiff base, Proc. Natl. Acad. Sci. U.S.A. 87 Ž1990. 1018. J. Tittor, D. Oesterhelt, E. Bambert, Inversion of proton translocation in bacteriorhodopsin mutants D85N, D85T, and D85,96N, Biophys. Chem. 67 Ž1994. 1682. M. Kotaoka, H. Kamikubo, F. Tokunaga, L.S. Brown, Y. Yamazaki, A. Maeda, M. Sheves, R. Needleman, J.K. Lanyi, Energy coupling in an ion pump. The reprotonation switch of bacteriorhodopsin, J. Mol. Biol. 243 Ž1994. 621. I. Rousso, N. Friedman, M. Sheves, M. Ottolenghi, p K a of the protonated Schiff base and aspartic-85 in the bacteriorhodopsin binding site is controlled by a specific geometry between two residues, Biochemistry 34 Ž1995. 12059. S. Druckmann, M. Ottolenghi, I. Rousso, N. Friedman, M. Sheves, Time-resolved titrations of the schiff base and of the Asp-85 residue in artificial bacteriorhodopsins, Biochemistry 34 Ž1995. 12066. E. Bamberg, J. Tittor, D. Oesterhelt, Light-driven proton or chloride pumping in halorhodopsin, Proc. Natl. Acad. Sci. U.S.A. 90 Ž1993. 12059. J. Tittor, U. Schweiger, D. Oesterhelt, E. Bamberg, Inversion of proton translocation in bacteriorhodopsin mutants D85N, D85T, and D85,96N, Biophys. J. 67 Ž1994. 1682. L.S. Brown, A.B. Druzhko, S.K. Chamorovsky, pH dependence of the adsorption spectra and photochemical transformation of the archaeorhodopsins, Biophysics 37 Ž1992. 66. G.T. Rikshireva, M.K. Pulatova, H.N. Chekuleva, A.B. Druzhko, V.A. Kositsyna, Letter: EPR study of bacteriorhodopsin-containing systems, Biofizika 20 Ž5. Ž1975. 938. A.B. Druzhko, S.K. Zharmukhamedov, N.N. Vsevolodov, Spectral transformations in purple membranes of Halobacterium halobium: effect of 560–570 transition and blue light on photochemical processes, Biofizika 23 Ž1978. 1019. A.B. Druzhko, S.K. Chamorovsky, The cycle of photochromic reaction of the bacteriorhodopsin analog with 4-ketoretinal, Biosystems 35 Ž1995. 133. A.B. Druzhko, H.H. Weetall, Photoinduced transformation of wildtype and D96N-mutated 4-keto-bacteriorhodopsin gelatin films, Thin Solid Films 293 Ž1997. 281. L.A. Drachev, A.D. Kaulen, V.P. Skulachev, Nature of the electrogenic phases of the bacteriorhodopsin photocycle and localization of retinal, Dokl. Akad. Nauk SSSR 281 Ž1985. 176. A.R. de Lera, B. Iglesias, J. Rodriguez, R. Alvarez, S. Lopez, X. Villanueva, F. Padros, Experimental and theoretical analysis of the steric tolerance of the binding site of bacterioopsin with the use of side-chain methyl-shifted retinal analogs, J. Am. Chem. Soc. 117 Ž1995. 8220. S. Subramaniam, T. Marti, S.J. Rosselet, K.J. Rothschild, H.G. Khorana, The reaction of hydroxylamine with bacteriorhodopsin studied with mutants that have altered photocycles: selective reactivity of different photointermediates, Proc. Natl. Acad. Sci. U.S.A. 88 Ž1991. 2583.
H.H. Weetall et al.r Bioelectrochemistry 51 (2000) 27–33 w23x N.V. Katre, P.K. Wolber, W. Stoeckenius, R.M. Stroud, Attachment siteŽs. of retinal in bacteriorhodopsin, Proc. Natl. Acad. Sci. U.S.A. 78 Ž1981. 4068. w24x B. Robertson, E.P. Lukashev, Rapid pH change due to bacteriorhodopsin measured with a tin oxide electrode, Biophys. J. 68 Ž1995. 1507.
33
w25x J. Le Coutre, J. Tittor, D. Oesterhel, K. Gerwert, Experimental evidence for hydrogen-bonded network proton transfer in bacteriorhodopsin shown by Fourier transform infrared spectroscopy using azide as catalyst, Proc. Natl. Acad. Sci. U.S.A. 92 Ž1995. 4962.
Measurement of proton release and uptake by analogs of bacteriorhodopsin Howard H. Weetall a,) , Anna Druzhko a , Angel R. de Lera b,1, Rosana Alvarez b,1, Baldwin Robertson a a b
Biotechnology DiÕision, National Institute of Standards and Technology, Gaithersburg, MD 20899, USA Department of Organic Chemistry, UniÕersity of Santiago de Compostela, Santiago de Compostela, Spain Received 25 March 1999; received in revised form 14 October 1999; accepted 17 October 1999
Abstract Proton release and subsequent uptake by several forms of bacteriorhodopsin ŽbR., including 4-keto analogs of wild-type ŽWT. and D96N and D85N mutants as well as the 9-demethylretinal analog of WT and D96N mutants, have been measured using a highly sensitive electrochemical technique. Release and uptake of protons by bR in membrane patches on a tin oxide electrode produce a current transient whose amplitude is proportional to the rate of pH change at the electrode surface. Profiles of proton release by the analogs vs. pH are substantially different from the profiles of the native proteins. q 2000 Elsevier Science S.A. All rights reserved. Keywords: Proton; Analog; Bacteriorhodopsin
1. Introduction Bacteriorhodopsin ŽbR. is a protein–chromophore complex that, when it absorbs yellow light, releases protons on the periplasmic side of the membrane and subsequently takes them up from the cytoplasmic side, thus pumping them across the membrane w1x. Retinal, the chromophore that is bound to Lys-216 of the protein by a protonated Schiff base, is responsible for the absorption that occurs at a maximum of approximately 570 nm. A quantum of yellow light induces a trans–cis isomerization of the retinal, followed by transitions of the protein–retinal complex through a number of intermediate states ŽI, J, K, L, M, N, and O. that are characterized by different protonation states of the Schiff base and of several amino acid residues within the protein w2,3x. The M state of the photocycle is formed when, as a result of the isomerization of retinal, the proton on the Schiff base is transferred to Asp-85, and subsequently a proton from another amino
)
Corresponding author. Tel.: q1-301-975-2000; fax: q1-301-5481087. E-mail address: [email protected] ŽH.H. Weetall.. 1 Present address: Universidad de Vigo, Departmento de Quimica Pura y Aplicada, Facultad de Ciencias, As Lagsas Marcosende, Vigo 36200, Spain.
acid XyŽ possibly Arg-82. w4x is released into the solution on the periplasmic side of the membrane and Asp-96, in turn, is reprotonated from the cytoplasmic solution. The proton release generally occurs within 50 ms after absorption of a quantum of light energy. The proton uptake in wild-type ŽWT. bR requires 10–20 ms. In D96N, the M state decays only when the Schiff base is reprotonated from solution, which can take over 100 s depending on pH w5–7x. In the next step, for both WT and D96N bR, retinal reisomerizes back to the all-trans configuration, and finally the proton from Asp-85 reprotonates the proton release group Xy, returning the complex to its initial state w2,3x. The D85N mutant, lacking the Asp in the 85 position, cannot accept a proton, so any release of a proton from the Schiff base must be by diffusion. This mutant shows the most dramatic pH dependence, because the pK of its Schiff base protonationrdeprotonation is approximately 6–8 w8,9x, compared with 13 observed for the WT w10–12x. Very little is known of the proton release function in this mutant. A study on purple membranes ŽPms. of D85N attached to a lipid bilayer w9x showed that a photocurrent can be generated in yellow light. The mechanism appears similar to that observed with halorhodopsin. Halorhodopsin can act as an inwardly directed proton pump after sequential absorption of a yellow and a blue photon w13x. This is due to a side reaction of the photocycle. At neutral pH, the
0302-4598r00r$ - see front matter q 2000 Elsevier Science S.A. All rights reserved. PII: S 0 3 0 2 - 4 5 9 8 Ž 9 9 . 0 0 0 7 2 - 0
28
H.H. Weetall et al.r Bioelectrochemistry 51 (2000) 27–33
Schiff base can deprotonate after absorption of a yellow photon, forming an M-like state. The proton is released on the cytoplasmic side. Blue light reprotonates the Schiff base from the extracellular side, completing the proton translocation. This translocation is amplified by azide, which increases the formation of the M-like state upon illumination with yellow light. For D85N, the Schiff base has a pK of around 8. In the dark at physiological pH, a portion of D85N has a deprotonated Schiff base with retinal in the all-trans configuration and a portion has it in the 13-cis configuration w14x. Absorption of a yellow photon and then a blue one produces inwardly directed proton transfer similar to that observed for halorhodopsin. The proton transfer of D85N, like that of halorhodopsin, is also enhanced by the addition of azide. Studies in this laboratory on D85N unoriented films indicate that at neutral pH, proton release occurs after exposure to blue light followed by yellow light. The measurement of proton release vs. pH by modified and unmodified forms of D96N and D85N can give important information on the mechanism of proton release by bR. Much effort has been made to characterize the 4-keto derivative of bR w13,15–19x. The transient electrical current that occurs when a photon is absorbed by 4-keto bR in air has been measured w20x. However, the specific charges whose displacement generates the current have not been identified. No measurement of the translocation of protons, as distinguished from other electrical charges, has been carried out. The intermediates of the photocycle of 4-keto WT bR have so far been identified only by their optical absorption spectra w15,18x. Even less is known about the 4-keto derivative of D85N. The mutant form of 9-demethylretinal has not been examined for photocycle activity or proton release. For 4-keto D96N and WT derivatives, the major optical characteristics are similar to those observed in gelatin films w19x. An initial absorbance peak at approximately 508 nm decays on exposure to yellow light and peaks appear at 390, 420 and 440 nm. These three peaks have been labeled as corresponding to M-like states w19x. Formation of the three peaks can occur in 60 ms for the 4-keto-WT, while in the 4-keto D96N, mutant in suspension requires several milliseconds w18x; they both require several milliseconds when the bR derivatives are in membrane patches in dried film w19x. Thus, release of protons by this derivative and the effect of pH on the release are of great interest. Since the characterization of retinal derivatives can be an important tool in understanding retinal–protein interactions, we have also chosen to examine the 9-demethylretinal derivatives of WT and D96N bR. The 9-demethyl analog has been described as binding to the apoprotein of bR with only minor structural changes w21x. Unlike the 4-keto analog, this analog has a modification along the backbone of the retinal chain rather than on one of the rings, where one would expect a greater effect. Informa-
tion related to proton release and uptake of this bR derivative will be helpful in determining the mechanism of action of both the 9-demethyl and the 4-keto derivatives.
2. Materials and methods Pms from the mutant and WT strains of Halobacterium salinarium were prepared by standard protocol w21x. An aqueous suspension of WT and D96N was illuminated by light at 500 nm in the presence of 0.5 M NH 3 OH, at pH 8.2–8.4 at 78C. In the case of the D85N mutant, which has a 20-fold lower reaction rate with NH 2 OH w22x, the apomembranes of this mutant were prepared differently. The suspension of the D85N mutant was illuminated by yellow light in the presence of 1 M NH 2 OH, 100 mM NaCl, and 25 mM Trizma base buffer, pH 8.6 at 228C. The activity of the apomembranes was determined by reconstruction with all-trans retinal ŽSigma, St. Louis, MO.. The 4-ketoretinal ŽFig. 1. was supplied by A.A. Khodonov ŽM.V. Lomonosov Institute of Fine Chemical Technology, Moscow, Russia.. For reconstruction, the analogs were first dissolved in isopropanol before adding to the apoprotein membranes in aqueous suspension. The 9-demethylretinal derivative ŽFig. 1. was prepared as previously described w23x. In several experiments, the retinal oxime was removed by extraction with bovine serum albumin as described by Katre et al. w23x before or after adding the retinal analog. This was carried out to determine the effect of retinal oxime on proton release and uptake. A film of membrane patches was prepared by depositing 10 ml of an aqueous suspension of membrane patches containing bR on an antimony-doped tin oxide-coated glass slide by placing the drop on the slide and letting the solution dry to form a spot approximately 6 mm " 1 mm in diameter. The concentration of the bR derivatives used for the pH studies varied from 0.1 to 1.0 mgrL.
Fig. 1. Structural formulas for the 9-demethylretinal Žtop. and the 4-ketoretinal Žbottom..
H.H. Weetall et al.r Bioelectrochemistry 51 (2000) 27–33
The tin oxide was used as the working electrode in an electrochemical cell ŽBAS CC-4 Flow Cell. modified to hold a working electrode 0.5 = 1.5 in.2 in size. A Lucite cover compressed the glass electrode and a Teflon gasket against the metal body of the cell, the latter serving as the counterelectrode. The Lucite cover was later replaced with a black nylon cover having a hole 5 mm in diameter directly over the 6 mm bR spot. The active area of the bR film was the spot on the tin oxide that was illuminated through the Lucite cover, or for later experiments, through the 5 mm hole in the black nylon cover. Using the opaque cover with the hole made the area more reproducible. The Teflon gasket was 0.25 mm thick and had a slot Žwith semicircular ends. cut in it that was 17 mm long and 4.5 mm wide. Thus, the walls of the cell were: tin oxide on the front where the light entered, the metal counterelectrode on the rear, and the Teflon on the bottom, top, and sides. By calculation, the volume of the flowcell was approximately 18 ml so that five drops washed the cell 15 times. Access to the reference electrode was through an approximately 0.2-mm hole in an insulating plug in the metal counterelectrode. The electrolyte was a mixture of three buffers: potassium citrate, potassium phosphate, and potassium borate, each 0.005 M in concentration plus 0.05 M potassium chloride. The pH of the solutions was adjusted with hydrochloric acid or potassium hydroxide. The reference electrode was silverrsilver chloride ŽBAS MW 2021.. The cell was connected to a potentiostat consisting of an operational amplifier ŽOPA111. with a 1 M V resistor in a feedback circuit. The voltage across the resistor was amplified by an instrumentation amplifier ŽAD524. with gain s 1. The amplifiers were battery-powered and completely enclosed along with the electrochemical cell in solid aluminum shielding to reduce electrical interference. The output was connected by coaxial cable to an oscilloscope ŽTektronix TDS 410., that could be set from 1.00 to 0.5 mVrbox, and 500 data points were collected in 2 s, so the response time for the system of approximately 4 ms. A horizontal beam of light from a mercury-arc lamp ŽLEP, HBO 100. was filtered by a 475-nm long pass filter ŽOriel 59490. and focused through a hole in the shielding onto the tin oxide electrode. The rest of the apparatus used were similar to that described previously w24x.
3. Results and discussion Under intense yellow light, retinal in the presence of excess hydroxylamine is photolyzed and reacts to form retinal oxime. Pms treated in this manner lose their color. When the chromophore analog is added, reconstitution of the bR molecule occurs, with the new chromophore attached to the protein and the retinal oxime still remaining but unattached. Since there is the possibility that in time an
29
exchange might occur between the natural retinal oxime and the analog bound to the opsin, reconverting the bR back to its natural state, any proton release or uptake observed might be attributable to reversion product. However, when the retinal oxime is removed from the opsin before introduction of the chromophore analog, there is no chance for reconversion to occur, and one can be confident that no natural chromophore bR is present. Measurement of the proton release and uptake of WT bR opsin after removal of the retinal oxime showed responses identical to that observed before removal of the unbound oxime. This response did not exceed 1 nA, which is about as small a current as we can observe. Thus, all other experiments were carried out without removing the retinal oxime, since its presence evidently had no effect. The proton release and uptake functions were measured by observing the amplitude of a light-induced current transient generated by the potentiostat connected to the electrochemical cell. The current rises because bR in the Pm patches on the tin oxide electrode releases protons near the electrode surface w24x. There are two physical effects that can result from motion of ions or charged complexes that occur when light is absorbed by bR. The first is a charge-displacement current and the second is a transient current due to the pH change that results from the release and uptake of protons. The voltage generated by bR on a tin oxide electrode in air is of the first kind; it is due to charge displacement and leads to an observed current w17x. However, this current is too small to be detected in our system. The current generated by the potentiostat when light excites the bR is of the second kind. It is proportional to the rate of change of the pH produced at the electrode surface by the protons released or taken up w23x. This provides a highly sensitive measurement of transient pH changes much more sensitive than can be achieved with a typical pH meter. However, the most important distinction between the two mechanisms is that measurement of the charge-displacement current does not reveal anything about just which ions or charged complexes are the ones that move and produce the current. Any charge that moves produces current. In contrast, the current observed using the tin oxide pH-sensitive electrode in the electrochemical cell is due specifically to release or uptake of protons. Both the proton-release transient that occurs when light is turned on and the proton-uptake transient that occurs when the light is turned off provide a direct measurement of stages of the proton-transport function of bR. Since the studies were carried out with randomly oriented patches, approximately half of the Pms has the cytoplasmic side toward the electrode and half has the periplasmic side toward the electrode. When the light is turned on, both release and uptake of protons occur near the electrode surface. However, there is a delay between the proton release and the subsequent uptake. Deprotonation requires 50 ms while reprotonation requires approximately 10 ms so the current due to proton release over-
30
H.H. Weetall et al.r Bioelectrochemistry 51 (2000) 27–33
whelms the current due to the slower uptake. When the light is turned off, there is only proton uptake and the current is in the opposite direction. We measured the amplitudes of photoinduced transient current generated by the 4-keto analogs of WT, D96N, and D85N. We chose to examine these because the mutations have profound effects when the chromophore is natural retinal. Asp-96 is the donor in the reprotonation of the Schiff base and Asp-85 is the acceptor in its deprotonation. We included the 9-demethylretinal because one might expect little change in the properties of this analog as compared to the 4-keto analog. This gives us some information on the effects attributable to changes in different parts of the chromophore since the 9-demethylretinal is modified on the polyene side chain and the 4-ketoretinal is modified on the ring structure ŽFig. 1.. We have chosen to examine proton release over a pH range of 3–9 because it is well-known that the pK of the Schiff base of several of the retinal analogs is smaller than for the natural chromophore for which the pK of the Schiff base is between 12 and 13 w10–12x. In the case of most of these analogs, the pK of the Schiff base evidently has been decreased to between 6 and 9 w10–12x. Studying pH profiles of both natural retinal bR and analog bR should give us greater insight into the mechanism of the proton release and uptake. The pH profiles of the 4-ketoretinal-WT, 4-ketoretinalD96N, 9-demethylretinal-WT, and the 9-demethylretinalD96N derivatives are shown in Fig. 2. Each of these derivatives shows a reversal in the proton release and uptake current transient at acid pH. This suggests that, as previously reported w10–12x, the pK of the Schiff base in the analogs must be lower. For the 9-demethylretinal derivative, deprotonation of the Schiff base has been eliminated or slowed to such a rate that the uptake completely dominates the release. It has also been suggested that the pK at the 85 position in similar analogs changes from 3.2 to above 9 w11x. Thus, the 85 position tends to remain protonated. For the 4-keto D85N, we could not observe any proton transfer under the conditions we examined. This may be because no more then 10% of the apoprotein was reconstituted with the 4-ketoretinal. The native form of D85N shows an M-like spectral peak in the dark because a fraction of the chromophore is in the all-trans configuration. Blue light causes decay of the M-like state and the return of the chromophore to the cis configuration. Turning on yellow light causes proton release as indicated by the appearance of a transient photocurrent whose direction corresponds to a decrease of pH at the electrode surface. Removal of the light causes an immediate proton uptake away from the electrode surface. Since the film is unoriented, the experiment does not show whether the uptake occurs on the cytoplasmic side and the release occurs on the extracellular side of the Pm. However, Tittor et al. w9x, using bilipid-oriented membrane vesicles, observed a transient photocurrent upon excitation
Fig. 2. The normalized pH profiles of the light-on transient current peaks for WT and mutant bR analog. Positive currents represent net proton release, negative currents represent net proton uptake. The current due to proton release was normalized by dividing by the current at the optimum pH for each derivative. The relative current at optimum pH for each analog was taken as 1.00. Open triangles represent 9-demethylretinal WT, closed circles represent 4-ketoretinal D96N, and open circles represent 4-ketoretinal WT. The inverted triangles represent 9-demethylretinal D96N. The buffer used in all studies was 0.005 M each of potassium citrate, potassium phosphate, potassium borate and 0.05 M potassium chloride with pH adjusted by adding hydrochloric acid or potassium hydroxide solutions.
with yellow light. They believe that the transient current was inwardly directed. This was determined on the assumption that the WT Pms and the Pms of the mutants absorbed preferentially onto the planar bilayer with the extracellular side toward the bilayer. The oriented lipid bilayers and the unoriented films appear to show similar results with yellow light. In both cases, one observes a transient photocurrent in the same direction, indicating proton release, followed by a second transient response in the opposite direction on removal of the light source. The D85N Pms in the lipid bilayer release protons on the cytoplasmic side w9x. In the unoriented films, we see proton release toward the electrode surface which is consistent with release of protons on the cytoplasmic side, since half of the patches is oriented with the cytoplasmic side toward the electrode. The side of the release will be confirmed in a planned study of oriented films. The natural-chromophore D85N bR photocurrent transient was examined as a function of pH. The pK of the Schiff base in D85N is approximately 8 w8,9x. The pH increase from 3 to 9 significantly shifts the equilibrium of the ground state and the M-like state towards the M-like
H.H. Weetall et al.r Bioelectrochemistry 51 (2000) 27–33
Fig. 3. The normalized pH profiles of light-on transient current peaks for bRs containing natural chromophore Žretinal.. Positive currents represent net proton release, negative currents represent net proton uptake. The current due to proton release was normalized by dividing the current at the optimum pH for each derivative. The relative current at optimum pH for each bR preparation was taken as 1.00. The X represents the WT, the plus represents D85N, and the closed triangle represents D96N. The buffer used in all studies was 0.005 M potassium citrate, potassium phosphate, potassium borate and 0.05 M potassium chloride with pH adjusted by adding hydrochloric acid or potassium hydroxide solutions.
state at higher pH. The normal rise of the M-like state is known to be blocked in D85N w8x. However, minor amounts of the intermediate are formed due to other mechanisms, including direct deprotonation to the medium. Alternatively, for pH close to the pK of the Schiff base, light
31
excitation may lower the pK and shift the equilibrium of the Schiff base towards deprotonation w8x. The presence of a photoinduced current at a pH between 5 and 8 suggests that this may be the case. The pH profiles of the 4-ketoretinal and the 9-demethylretinal derivatives of the D96N look very similar ŽFig. 2.. The differences in the retinal substitution appear to play less of a role in determining the pH characteristics of these derivatives than does the amino acid substitution. When comparing the natural-chromophore D96N with the two analogs, little difference is observed ŽFig. 2.. If one examines the pH transient current profiles for bRs containing the native chromophore ŽFig. 3., one observes smaller reversals of the current transient for both WT and D96N at acid pH ŽFig. 3.. As previously mentioned for D96N, there is a smaller difference between the pH profiles of the 4-ketoretinal analog and the 9-demethylretinal analog and the native form. This suggests that the absence of Asp-96 plays a far more important role in determining the proton uptake and release mechanism then does the chromophore. The 9-demethylretinal analogs were added to this study because the removal of the methyl group on the polyene portion of the retinal should have a small effect, compared to the 4-ketoretinal analog, where substitution is made on the ring structure. The pH profiles of the analogs of the WT derivatives showed greater reversal of the light-induced current transient for pH lower than observed for the D96N analogs. This suggests that in the WT, the chromophore plays a larger role in determining the characteristics of proton release and uptake than in the D96N analogs. Computational studies w21x suggest that the location of the methyl groups on the polyene side chain determines the overall shape of the retinal ligand. According to the suggested model w14x, the effect of the methyl group should be purely steric in nature. It is suggested that Trp-182 contacts the C9-methyl group, and that this contact is critical for the photocycle function. Our data suggest that the effects of the removal of the 9-methyl functional-
Table 1 Sample
pH optimum
Peak current a mean ŽnA.
Peak current standard deviation ŽnA.
WT WT 4-keto WT 9-demethyl D96N D96N 4-keto D96N 9-demethyl D85N D85N 4-keto
7.0 6.5 6.5 6.5 6.5 6.0 7.0 Not detectable
220 23 116 400 29 74 18 Not detectable
12 2 6 18 2 5 3 Not detectable
a The values shown here represent the light on current density transient only. The peak currents were determined at the optimum pH for proton release. Samples were irradiated with blue light for 1 min before a 1-s flash of yellow light. All samples contained 0.5 mgrml of bR in concentration. Sample size was 20 ml. The sample labeled NA did not show detectable proton release or uptake. The standard deviations of the peak current values are for five replicates on the same electrode. Each sample film was characterized on separate electrodes. The uncertainty for bR on different electrodes is 20% of the peak currents.
32
H.H. Weetall et al.r Bioelectrochemistry 51 (2000) 27–33
ity may have a larger effect on the protein than computational studies anticipate. We have also compared the peak values for the proton release current transients for each of the bRs at equal protein concentrations ŽTable 1.. The results show that the greatest magnitude was observed with D96N while the smallest value was observed with D85N. These data are contrary to that of Le Coutre et al. w25x. These studies using D pH measurements in bR liposomes showed that D96N had ) 3% of the proton pumping activity of the WT. However, the peak current for proton release depends on the turnover rate and since the total turnover time of the WT photocyle is short in comparison with the time scale of the current transient. Since proton uptake begins earlier when the turnover is rapid, this diminishes the net magnitude of the observed current transient peak. In the case of the slower mutants and analogs, the peak current transients are more closely related to the ground to M-state transition efficiency because the M-to-ground state transition is much slower than the time scale of the peak current transient. As previously stated, the current transient is directly related to the rate of proton transport. We have examined and compared the proton uptake and release of WT and two mutant bRs containing both native and artificial chromophores. Substitution of either 4-ketoretinal or 9-demethylretinal for the native chromophore substantially reduces the proton transport by WT at acid pH, but only slightly reduces the proton release by D96N.
w5x
w6x
w7x
w8x
w9x
w10x
w11x
w12x
w13x
w14x
Acknowledgements w15x
This work was supported by the National Institute of Standards and Technology. The work also received financial support from FIS Žcontract 95r1534., the Xunta de Galicia Žgrant XUGA20904B95., and the University of Santiago de Compostela Žfellowship to R. Alvarez.. The authors wish to thank Dr. Tonya Herne from the National Institute of Standards and Technology for her valuable suggestions while reviewing this manuscript.
References w1x D. Oesterhelt, W. Stockenius, Rhodopsin-like protein from the purple membrane of Halobacterium halobium, Nat. New Biol. 233 Ž1971. 149. w2x M. Kono, S. Misra, T.G. Ebrey, pH dependence of light-induced proton release by bacteriorhodopsin, in: M.B. Jackson ŽEd.., Thermodynamics of Membrane Receptors and Channels, CRC Press, Cleveland, 1993, p. 353. w3x R.A. Mathies, S.W. Lin, J.B. Ames, W.T. Pollard, From femtoseconds to biology: mechanism of bacteriorhodopsin’s light-driven proton pump, Annu. Rev. Biophys. Biophys. Chem. 20 Ž1991. 491. w4x S.P. Balashov, R. Govindjee, M. Kono, E. Imasheva, E. Lukashev, T.G. Ebrey, R.K. Crouch, D.R. Menick, Y. Feng, Effect of the arginine-82 to alanine mutation in bacteriorhodopsin on dark adapta-
w16x
w17x
w18x
w19x
w20x
w21x
w22x
tion proton release and the photochemical cycle, Biochemistry 32 Ž1993. 10331. A. Miller, D. Oesterhelt, A guide to electron paramagnetic resonance spectroscopy of photosystem II membranes, Biochim. Biophys. Acta 1020 Ž1990. 57. M. Holz, L.A. Drachev, T. Mogi, H. Otto, A.D. Kaulen, M.P. Heyn, V.P. Skulachev, H.G. Khorana, Replacement of aspartic acid-96 by asparagine in bacteriorhodopsin slows both the decay of the M intermediate and the associated proton movement, Proc. Natl. Acad. Sci. U.S.A. 86 Ž1989. 2167. J. Tittor, C. Soell, D. Oesterhelt, H.-J. Butt, E. Bamberg, A defective proton pump, point-mutated bacteriorhodopsin Asp-96 . . . Asn is fully reactivated by azide, EMBO J. 8 Ž1989. 3477. H. Otto, T. Marti, M. Holz, T. Mogi, L.J. Stern, F. Engel, H.G. Khorana, M.P. Heyn, Substitution of amino acid Asp-85, Asp-212, and Arg-82 in bacteriorhodopsin affects the proton release of the pump and the pK of the Schiff base, Proc. Natl. Acad. Sci. U.S.A. 87 Ž1990. 1018. J. Tittor, D. Oesterhelt, E. Bambert, Inversion of proton translocation in bacteriorhodopsin mutants D85N, D85T, and D85,96N, Biophys. Chem. 67 Ž1994. 1682. M. Kotaoka, H. Kamikubo, F. Tokunaga, L.S. Brown, Y. Yamazaki, A. Maeda, M. Sheves, R. Needleman, J.K. Lanyi, Energy coupling in an ion pump. The reprotonation switch of bacteriorhodopsin, J. Mol. Biol. 243 Ž1994. 621. I. Rousso, N. Friedman, M. Sheves, M. Ottolenghi, p K a of the protonated Schiff base and aspartic-85 in the bacteriorhodopsin binding site is controlled by a specific geometry between two residues, Biochemistry 34 Ž1995. 12059. S. Druckmann, M. Ottolenghi, I. Rousso, N. Friedman, M. Sheves, Time-resolved titrations of the schiff base and of the Asp-85 residue in artificial bacteriorhodopsins, Biochemistry 34 Ž1995. 12066. E. Bamberg, J. Tittor, D. Oesterhelt, Light-driven proton or chloride pumping in halorhodopsin, Proc. Natl. Acad. Sci. U.S.A. 90 Ž1993. 12059. J. Tittor, U. Schweiger, D. Oesterhelt, E. Bamberg, Inversion of proton translocation in bacteriorhodopsin mutants D85N, D85T, and D85,96N, Biophys. J. 67 Ž1994. 1682. L.S. Brown, A.B. Druzhko, S.K. Chamorovsky, pH dependence of the adsorption spectra and photochemical transformation of the archaeorhodopsins, Biophysics 37 Ž1992. 66. G.T. Rikshireva, M.K. Pulatova, H.N. Chekuleva, A.B. Druzhko, V.A. Kositsyna, Letter: EPR study of bacteriorhodopsin-containing systems, Biofizika 20 Ž5. Ž1975. 938. A.B. Druzhko, S.K. Zharmukhamedov, N.N. Vsevolodov, Spectral transformations in purple membranes of Halobacterium halobium: effect of 560–570 transition and blue light on photochemical processes, Biofizika 23 Ž1978. 1019. A.B. Druzhko, S.K. Chamorovsky, The cycle of photochromic reaction of the bacteriorhodopsin analog with 4-ketoretinal, Biosystems 35 Ž1995. 133. A.B. Druzhko, H.H. Weetall, Photoinduced transformation of wildtype and D96N-mutated 4-keto-bacteriorhodopsin gelatin films, Thin Solid Films 293 Ž1997. 281. L.A. Drachev, A.D. Kaulen, V.P. Skulachev, Nature of the electrogenic phases of the bacteriorhodopsin photocycle and localization of retinal, Dokl. Akad. Nauk SSSR 281 Ž1985. 176. A.R. de Lera, B. Iglesias, J. Rodriguez, R. Alvarez, S. Lopez, X. Villanueva, F. Padros, Experimental and theoretical analysis of the steric tolerance of the binding site of bacterioopsin with the use of side-chain methyl-shifted retinal analogs, J. Am. Chem. Soc. 117 Ž1995. 8220. S. Subramaniam, T. Marti, S.J. Rosselet, K.J. Rothschild, H.G. Khorana, The reaction of hydroxylamine with bacteriorhodopsin studied with mutants that have altered photocycles: selective reactivity of different photointermediates, Proc. Natl. Acad. Sci. U.S.A. 88 Ž1991. 2583.
H.H. Weetall et al.r Bioelectrochemistry 51 (2000) 27–33 w23x N.V. Katre, P.K. Wolber, W. Stoeckenius, R.M. Stroud, Attachment siteŽs. of retinal in bacteriorhodopsin, Proc. Natl. Acad. Sci. U.S.A. 78 Ž1981. 4068. w24x B. Robertson, E.P. Lukashev, Rapid pH change due to bacteriorhodopsin measured with a tin oxide electrode, Biophys. J. 68 Ž1995. 1507.
33
w25x J. Le Coutre, J. Tittor, D. Oesterhel, K. Gerwert, Experimental evidence for hydrogen-bonded network proton transfer in bacteriorhodopsin shown by Fourier transform infrared spectroscopy using azide as catalyst, Proc. Natl. Acad. Sci. U.S.A. 92 Ž1995. 4962.