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Deactivation and Proton Transfer in Light-induced Metarhodopsin II/Metarhodopsin III Conversion

2007; Elsevier BV; Volume: 282; Issue: 14 Linguagem: Inglês

10.1074/jbc.m610658200

1083-351X

Eglof Ritter, Matthias Elgeti, Klaus Peter Hofmann, Franz Bartl,

Retinal Development and Disorders

Vertebrate rhodopsin shares with other retinal proteins the 11-cis-retinal chromophore and the light-induced 11-cis/trans isomerization triggering its activation pathway. However, only in rhodopsin the retinylidene Schiff base bond to the apoprotein is eventually hydrolyzed, making a complex regeneration pathway necessary. Metabolic regeneration cannot be short-cut, and light absorption in the active metarhodopsin (Meta) II intermediate causes anti/syn isomerization around the retinylidene linkage rather than reversed trans/cis isomerization. A new deactivating pathway is thereby triggered, which ends in the Meta III “retinal storage” product. Using time-resolved Fourier transform infrared spectroscopy, we show that the identified steps of receptor activation, including Schiff base deprotonation, protein structural changes, and proton uptake by the apoprotein, are all reversed. However, Schiff base reprotonation is much faster than the activating deprotonation, whereas the protein structural changes are slower. The final proton release occurs with pK ≈ 4.5, similar to the pK of a free Glu residue and to the pK at which the isolated opsin apoprotein becomes active. A forced deprotonation, equivalent to the forced protonation in the activating pathway, which occurs against the unfavorable pH of the medium, is not observed. This explains properties of the final Meta III product, which displays much higher residual activity and is less stable than rhodopsin arising from regeneration with 11-cis-retinal. We propose that the anti/syn conversion can only induce a fast reorientation and distance change of the Schiff base but fails to build up the full set of dark ground state constraints, presumably involving the Glu134/Arg135 cluster. Vertebrate rhodopsin shares with other retinal proteins the 11-cis-retinal chromophore and the light-induced 11-cis/trans isomerization triggering its activation pathway. However, only in rhodopsin the retinylidene Schiff base bond to the apoprotein is eventually hydrolyzed, making a complex regeneration pathway necessary. Metabolic regeneration cannot be short-cut, and light absorption in the active metarhodopsin (Meta) II intermediate causes anti/syn isomerization around the retinylidene linkage rather than reversed trans/cis isomerization. A new deactivating pathway is thereby triggered, which ends in the Meta III “retinal storage” product. Using time-resolved Fourier transform infrared spectroscopy, we show that the identified steps of receptor activation, including Schiff base deprotonation, protein structural changes, and proton uptake by the apoprotein, are all reversed. However, Schiff base reprotonation is much faster than the activating deprotonation, whereas the protein structural changes are slower. The final proton release occurs with pK ≈ 4.5, similar to the pK of a free Glu residue and to the pK at which the isolated opsin apoprotein becomes active. A forced deprotonation, equivalent to the forced protonation in the activating pathway, which occurs against the unfavorable pH of the medium, is not observed. This explains properties of the final Meta III product, which displays much higher residual activity and is less stable than rhodopsin arising from regeneration with 11-cis-retinal. We propose that the anti/syn conversion can only induce a fast reorientation and distance change of the Schiff base but fails to build up the full set of dark ground state constraints, presumably involving the Glu134/Arg135 cluster. The photoreceptor rhodopsin located in the retinal rods of the vertebrate eye contains the chromophore 11-cis-retinal bound by a protonated Schiff base to Lys296 of the apoprotein (1Hargrave P.A. McDowell J.H. FASEB J. 1992; 6: 2323-2331Crossref PubMed Scopus (234) Google Scholar). Light absorption triggers isomerization around the C11=C12 double bond of the polyene chain of the chromophore (2Hubbard R. Wald G. Science. 1952; 115: 60-63Crossref Scopus (12) Google Scholar, 3Hubbard R. Wald G. J. Gen. Physiol. 1952; 36: 269-315Crossref PubMed Scopus (235) Google Scholar, 4Yoshizawa T. Wald G. Nature. 1963; 197: 1279-1286Crossref PubMed Scopus (380) Google Scholar), leading to the strained all-trans-form and storage of two thirds of the light energy in the chromophore-protein system (5Wald G. Brown P.K. J. Gen. Physiol. 1953; 37: 189-200Crossref PubMed Scopus (292) Google Scholar, 6Kropf A. Vision Res. 1967; 7: 811-818Crossref PubMed Scopus (38) Google Scholar, 7Cooper A. Nature. 1979; 282: 531-533Crossref PubMed Scopus (181) Google Scholar, 8Kim J.E. Tauber M.J. Mathies R.A. Biochemistry. 2001; 40: 13774-13778Crossref PubMed Scopus (149) Google Scholar). The receptor subsequently proceeds through a number of intermediates each characterized by its specific absorption spectrum in the UV-visible and mid infrared range. Related conformational changes of the binding pocket and of other, more remote parts of the apoprotein eventually lead to the active G-protein binding state, metarhodopsin II (Meta II). 3The abbreviations used are: Meta, metarhodopsin; FTIR, Fourier transform infrared; R-/RR-Meta, intermediates of Meta II photolysis; BTP, 1,3-bis-(tris(hydroxymethyl)-methyl-amino)propane; LED, light-emitting diode; Nd:YAG, neodymium yttrium aluminum garnet. It is in equilibrium with its precursor metarhodopsin I (Meta I), depending on temperature and pH (9Matthews R.G. Hubbard R. Brown P.K. Wald G. J. Gen. 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The formation of the active species through the photointermediates has been described as a stepwise lowering of the stabilizing effect of the Schiff base counterion, which is a complex structure that comprises highly conserved Glu181 and Glu113. In Meta I, the counterion appears to undergo a shift relative to the Schiff base, whereas the receptor is still inactive (16Ludeke S. Beck M. Yan E.C. Sakmar T.P. Siebert F. Vogel R. J. Mol. Biol. 2005; 353: 345-356Crossref PubMed Scopus (97) Google Scholar). The subsequent deprotonation of the Schiff base linkage between Lys296 and the aldehyde group of the retinal is reflected in the strong shift of the absorption maximum from 480 to 380 nm, which is the spectral signature of Meta II and results in a protonation of Glu113 (17Jäger F. Fahmy K. Sakmar T.P. Siebert F. Biochemistry. 1994; 33: 10878-10882Crossref PubMed Scopus (146) Google Scholar). Proton uptake to Meta II must occur in a spectrally silent conversion forming a separate product Meta IIb (18Arnis S. Hofmann K.P. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 7849-7853Crossref PubMed Scopus (177) Google Scholar). This step involves the residue Glu134, which is part of a cluster that stabilizes the apoprotein in the dark (19Arnis S. Fahmy K. Hofmann K.P. Sakmar T.P. J. Biol. Chem. 1994; 269: 23879-23881Abstract Full Text PDF PubMed Google Scholar, 20Arnis S. Hofmann K.P. Biochemistry. 1995; 34: 9333-9340Crossref PubMed Scopus (54) Google Scholar, 21Fahmy K. Sakmar T.P. Siebert F. Biochemistry. 2000; 39: 10607-10612Crossref PubMed Scopus (74) Google Scholar). Under physiological conditions the pH value of the surrounding medium is higher than the intrinsic pK of the uptake group of the apoprotein (estimated pK ∼ 4 (22Vogel R. Siebert F. J. Biol. Chem. 2001; 276: 38487-38493Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar)), so that the light energy stored in the chromophore is partly used to enforce this protonation step and to shift the pK of the proton uptake group into the neutral range (23Hofmann K.P. Ernst O.P. Zeitschrift. für. Medizinische. Physik. 2001; 11: 217-225Crossref PubMed Google Scholar, 24Buczylko J. Saari J.C. Crouch R.K. Palczewski K. J. Biol. Chem. 1996; 271: 20621-20630Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). This “forced protonation” occurs as long as all-trans-retinal has all structural determinants, including the 9-methyl group and an intact β-ionone ring enabling it to act as a rigid scaffold (25Vogel R. Fan G.B. Sheves M. Siebert F. Biochemistry. 2000; 39: 8895-8908Crossref PubMed Scopus (65) Google Scholar, 26Meyer C.K. Böhme M. Ockenfels A. Gärtner W. Hofmann K.P. Ernst O.P. J. Biol. Chem. 2000; 275: 19713-19718Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). In rhodopsin regenerated with 9-demethyl-retinal or 11-cis-acyclic retinal (27Bartl F.J. Fritze O. Ritter E. Herrmann R. Kuksa V. Palczewski K. Hofmann K.P. Ernst O.P. J. Biol. Chem. 2005; 280: 34259-34267Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar, 28Vogel R. Siebert F. Ludeke S. Hirshfeld A. Sheves M. Biochemistry. 2005; 44: 11684-11699Crossref PubMed Scopus (45) Google Scholar) light-induced isomerization of the chromophore leads only to an active conformation when a low pH of the bulk phase supports proton uptake by the apoprotein. This can be interpreted in terms of an incomplete scaffold function of the retinal in these modified pigments. Active Meta II eventually decays in opsin and all-trans-retinal (29Wald G. Science. 1968; 162: 230-239Crossref PubMed Scopus (804) Google Scholar). For completion of the visual cycle, fresh 11-cis-retinal has to be supplied by a complex retinoid cycle to regenerate rhodopsin. This is remarkable because other retinal proteins such as archaeal or invertebrate rhodopsins can simply regenerate the ground state when a second photon in the active state is absorbed (for review, see Refs. 30Gärtner W. Stavenga D.G. DeGrip W.J. Pugh E.N.J. Molecular Mechanism in Visual Transduction. Vol. 3. Elsevier, Amsterdam2000: 297-388Google Scholar and 31Lanyi J.K. Acta Physiol. Scand. Suppl. 1992; 607: 245-248PubMed Google Scholar). Spectroscopic data show that blue light absorption of the active Meta II state does not lead back to the ground state (λmax = 500 nm) to a significant extent but to a product with an absorption maximum at 475 nm (32Bartl F.J. Ritter E. Hofmann K.P. J. Biol. Chem. 2001; 276: 30161-30166Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). By UV-visible and FTIR difference spectroscopy, this blue light-induced photoproduct of Meta II could be identified as Meta III (33Ritter E. Zimmermann K. Heck M. Hofmann K.P. Bartl F.J. J. Biol. Chem. 2004; 279: 48102-48111Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar, 34Zimmermann K. Ritter E. Bartl F.J. Hofmann K.P. Heck M. J. Biol. Chem. 2004; 279: 48112-48119Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar), a species already known as a side product of the thermal Meta II decay (9Matthews R.G. Hubbard R. Brown P.K. Wald G. J. Gen. Physiol. 1963; 47: 215-240Crossref PubMed Scopus (432) Google Scholar). Recent work has shown that this thermal product contains the chromophore in the Schiff base all-trans-15-syn configuration (35Vogel R. Siebert F. Mathias G. Tavan P. Fan G. Sheves M. Biochemistry. 2003; 42: 9863-9874Crossref PubMed Scopus (45) Google Scholar) and that the Meta III formed by light absorption is the same by all available criteria. Hence, Meta III is also triggered by light-induced anti/syn isomerization of the C=N double bond of the Schiff base (36Bartl F.J. Vogel R. Phys. Chem. Chem. Phys. 2007; 10 (1039/b616365c)Google Scholar). These findings substantiated the previous concept of the first and second switch (32Bartl F.J. Ritter E. Hofmann K.P. J. Biol. Chem. 2001; 276: 30161-30166Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar), which are now identified as the cis/trans isomerization of the polyene chain and the syn/anti isomerization of the Schiff base, respectively. The end product of the new light-induced pathway, Meta III, can form up to 80%, depending on the conditions, and has remarkable properties. Because of its long lifetime (up to hours (34Zimmermann K. Ritter E. Bartl F.J. Hofmann K.P. Heck M. J. Biol. Chem. 2004; 279: 48112-48119Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar)), it excludes the chromophore very efficiently from the regeneration pathway, which has led to the concept of a retinal storage form (34Zimmermann K. Ritter E. Bartl F.J. Hofmann K.P. Heck M. J. Biol. Chem. 2004; 279: 48112-48119Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar, 37Heck M. Schädel S.A. Maretzki D. Bartl F.J. Ritter E. Palczewski K. Hofmann K.P. J. Biol. Chem. 2003; 278: 3162-3169Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar, 38Schädel S.A. Heck M. Maretzki D. Filipek S. Teller D.C. Palczewski K. Hofmann K.P. J. Biol. Chem. 2003; 278: 24896-24903Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). Remarkably, the lifetime of Meta III depends on the presence of G-protein (34Zimmermann K. Ritter E. Bartl F.J. Hofmann K.P. Heck M. J. Biol. Chem. 2004; 279: 48112-48119Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar) and arrestin (39Sommer M.E. Smith W.C. Farrens D.L. J. Biol. Chem. 2005; 280: 6861-6871Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar, 40Sommer M.E. Smith W.C. Farrens D.L. J. Biol. Chem. 2006; 281: 9407-9417Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar), which was interpreted as an “inverse catalysis” of receptor conversion by G-protein. Given the fact that these proteins undergo light-dependent transport, and that the rate of rhodopsin regeneration is affected by the decay of Meta II and Meta III in contrast to the phosphorylation rate (41Paulsen R. Miller J.A. Brodie A.E. Bownds M.D. Vision Res. 1975; 15: 1325-1332Crossref PubMed Scopus (23) Google Scholar), interesting possibilities of regulation are thereby opened. The putative physiological implications and mechanistic significance of the light-induced deactivation pathway, which might play a role under conditions of bright light illumination, have led us to investigate its kinetics and the intermediates involved. Time-resolved UV-visible spectroscopy and electrical measurements of the activating and the deactivating pathway have already given indications that the light-induced absorption change related to Schiff base reprotonation occurs at a surprisingly high rate, namely even faster than the deprotonation linked to Meta II formation (20Arnis S. Hofmann K.P. Biochemistry. 1995; 34: 9333-9340Crossref PubMed Scopus (54) Google Scholar, 33Ritter E. Zimmermann K. Heck M. Hofmann K.P. Bartl F.J. J. Biol. Chem. 2004; 279: 48102-48111Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar, 42Dickopf S. Mielke T. Heyn M.P. Biochemistry. 1998; 37: 16888-16897Crossref PubMed Scopus (26) Google Scholar). We have therefore attempted to obtain additional information on the kinetics of the conformational conversions by time-resolved infrared difference spectroscopy, which allows us to follow a variety of structural alterations of the protein connected to its activity occurring on a 10-ms time scale. It turns out that anti/syn isomerization causes proper reprotonation of the retinal Schiff base but fails to couple to structural changes that are mandatory to fully deactivate the protein. Rhodopsin Purification and Preparation—Rhodopsin in washed disk membranes was purified from fresh dark-adapted bovine retinae. In a first step, rod outer segments were prepared by a discontinuous sucrose gradient method (43Papermaster D.S. Methods Enzymol. 1982; 81: 48-52Crossref PubMed Scopus (258) Google Scholar). Subsequently, washed disk membranes were obtained by repetitive washes with a low ionic strength buffer and fatty acid free bovine serum albumin (44Sachs K. Maretzki D. Meyer C.K. Hofmann K.P. J. Biol. Chem. 2000; 275: 6189-6194Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar, 45Sachs K. Maretzki D. Hofmann K.P. Methods Enzymol. 2000; 315: 238-251Crossref PubMed Google Scholar). Rhodopsin membrane suspension was stored at -80 °C until use. As buffer, we used 1,3-bis(tris(hydroxymethyl)methylamino)propane (BTP), and pH values were adjusted with diluted (0.1 m) NaOH and HCl. During all preparation steps the sample was kept under dim red light (λ > 640 nm). FTIR Measurements—FTIR samples were prepared by a centrifugation procedure as described (32Bartl F.J. Ritter E. Hofmann K.P. J. Biol. Chem. 2001; 276: 30161-30166Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). Rhodopsin membrane suspension was centrifuged (30 min, 100,000 × g, 4 °C) to obtain a 2-3 mm pellet, which was exposed to dry air for 60 s to further reduce water content. Subsequently the pellet was transferred into a temperature-controlled cuvette consisting of two BaF2 windows and a 3-μm polytetrafluoroethylene spacer. FTIR spectra were measured in a Bruker IFS66v/s spectrometer equipped with a liquid nitrogen-cooled mercury cadmium telluride detector (J15D-series, EG&G Judson). Before measurement, each sample was equilibrated for at least 30 min in the spectrometer. FTIR spectra were recorded before and after illumination, and FTIR difference spectra were calculated by subtracting the spectra of the initial state (A) from the spectra of the final state (B) and termed as “B minus A difference spectra.” Illumination was performed with the following light sources: For non-time-resolved measurements, we used a ring of six green or blue LEDs placed directly at the cuvette (520 ± 10 nm or 400 ± 10 nm, respectively). With LEDs, the sample was illuminated for 4 s. For illumination during the time-resolved experiments, we used two different lasers: For activation of rhodopsin a Nd:YAG laser (Spectron Lasers SL 282G), tuned to 532 nm, was directly focused on the sample. For deactivation of Meta II, a second Nd:YAG laser (SL 456-10), emitting at 355 nm, was used to pump a customized dye-laser emitting at 389 nm (Dye: Exalite 389, Radiant Dyes, Germany). The pulse duration of the Nd:YAG was 6 ns. One flash activated or deactivated ∼40% of the rhodopsin on a sample spot of 5-mm diameter as estimated by bleaching the samples with LEDs of the appropriate wavelength. A complete conversion to Meta II or Meta III, respectively, could not be achieved by the flash illumination due to the absorption of a second photon by early intermediates of the reaction pathways. This induces back and/or side reactions to rhodopsin and isorhodopsin (activation) (9Matthews R.G. Hubbard R. Brown P.K. Wald G. J. Gen. Physiol. 1963; 47: 215-240Crossref PubMed Scopus (432) Google Scholar) or to so far unknown products (deactivation). To investigate whether a certain intermediate is active toward the G-protein, the “Extra-Meta II” assay was used (46Bartl F. Ritter E. Hofmann K.P. FEBS Lett. 2000; 473: 259-264Crossref PubMed Scopus (32) Google Scholar). A rhodopsin pellet was resuspended in 50 μl of a solution of BTP and 10 mm G-protein-derived high affinity peptide (Gtα-(340-350) VLEDLKSCGLF) (46Bartl F. Ritter E. Hofmann K.P. FEBS Lett. 2000; 473: 259-264Crossref PubMed Scopus (32) Google Scholar). This solution was centrifuged for 30 min at 100,000 × g and 4 °C. By this procedure, a pellet containing the peptide in excess was obtained. Time-resolved FTIR difference spectra were recorded with the rapid scan technique. To enhance time resolution, double-sided forward-backward measured interferograms were split into four single-sided interferograms. Because one scan takes 100.0 ms at a spectral resolution of 4 cm-1 and 64.8 ms at a spectral resolution of 8 cm-1, respectively, we achieved a time resolution of 25 ms at 4 cm-1 and 16.2 ms at 8 cm-1. Due to the fast reaction, activation was measured with both resolutions, 4 and 8 cm-1. The slower kinetics of deactivation allowed us to measure this reaction with the slower time resolution of 25 ms but with high spectral resolution of 4 cm-1. Sample excitation by the laser flash was synchronized with the forward signal of the mirror movement to minimize the influence of the flash on the signal. Each experiment was reproduced at least six times. Non-time-resolved measurements were done by averaging 128 scans; for time-resolved spectroscopy four independent measurements were averaged. Time courses of single bands were smoothed by a digital band-block filter and fitted to first order exponentials to evaluate the half times t½. The three-dimensional plots were smoothed by using the Savatzky-Golay algorithm. UV-visible Measurements—For measurements in the UVvisible spectral range, the same centrifugation procedure as described for FTIR was applied. The rhodopsin pellets were transferred to a temperature-controlled transmission cuvette consisting of two BaF2 windows and a 50-μm spacer of polytetrafluoroethylene. Measurements were performed with an OLIS RSM-16 spectrometer equipped with a photomultiplier tube as detector. Time resolution was 1 ms, and the total data collection time was 3 s. Flash illumination of the sample was applied using a flash lamp (Rapp OptoElectronic GmbH) with 2-ms flash duration. For illumination of rhodopsin and Meta II, a 480 nm shortwave cut-off (Schott GG 475) and a 400-450 nm band pass filter were used, respectively. During the flash, the detector of the spectrometer was covered by a shutter to avoid any influence of the flash light on the detector signal. For the decay measurement in Fig. 4B we used a Varian Cary 50 spectrophotometer scanning every 12 s over 3.5 min. In Fig. 1, we show the UV-visible spectra of the light-induced deactivation of Meta II and of the light-induced activation of rhodopsin for comparison, initiated by a blue and a green flash, respectively. The black line in Fig. 1 with an absorption maximum of 498 nm represents the spectrum of the ground state. At pH 6.0 and 10 °C, rhodopsin was activated by a green flash (λ > 480 nm, 2 ms). Spectra were recorded on a millisecond time scale and plotted every 20 ms (thin green lines). The first spectrum measured immediately after the flash shows an absorption maximum of 480 nm, indicative for the Meta I intermediate. Subsequently, this band vanishes while a new band at 380 nm appears, which reflects the formation of Meta II. A single exponential fit to the time course (trace a in the inset of Fig. 1) yields t½ = 70 ms. We achieved a maximum bleaching of ∼40% of total rhodopsin by one flash as described under “Experimental Procedures.” Quantitative conversion to Meta II was subsequently performed by bleaching the sample with green light (λ > 500 nm) for 10 s (Fig. 1, thick green line). A blue flash (400-450 nm, 2 ms) was then applied to induce deactivation of Meta II. The blue lines in Fig. 1 show the respective UV-visible spectra recorded on a millisecond time scale. Traces were plotted every 20 ms. The blue flash induces an immediate reduction of the intensity of the 380 nm absorption (half time, t½ < 5 ms) and a corresponding rise at ∼475 nm. Neither any significant further reduction of the 380 nm band nor any other notable change of the absorption spectra could be detected within a time scale comparable to the activation process. ∼40% of total Meta II were converted by the flash and quantitative conversion to the 475 nm product (dashed blue line) was subsequently achieved by continuous (10 s) blue light illumination of the sample. The absorption change at 380 nm evoked by the blue light flash is shown in trace b in the inset of Fig. 1 (blue). The fast kinetics cannot be resolved with the rapid scanning UV-visible spectrometer; the same experiment performed with a flash photolysis setup with microsecond time resolution (32Bartl F.J. Ritter E. Hofmann K.P. J. Biol. Chem. 2001; 276: 30161-30166Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar) yielded a half time of 4.5 ms at 10 °C for this process. These experiments indicate a fast, light-induced reprotonation of the Schiff base, which is faster than the deprotonation step in the activating pathway. Because the absorption maxima in the UV-visible spectra solely report on the protonation state of the chromophore, time-resolved FTIR difference spectroscopy was used to compare the kinetics of conformational changes of the apoprotein. Activation—Fig. 2A shows the FTIR difference spectra of rhodopsin illuminated by a 6 ns laser flash (532 nm, Nd:YAG) as a function of time in a three-dimensional plot. To demonstrate that the formation of both species, Meta I and Meta II, can be fully resolved with our experimental setup, the spectra were recorded at 0 °C and pH 6 every 25 ms. Under these conditions, the first spectrum after the flash is a Meta I minus rhodopsin difference spectrum, as indicated by the bands at 951/970 cm-1, the typical fingerprint approximately 1238/1205 cm-1, the doublet at 1536/1549 cm-1, and the characteristic band pattern approximately 1700 cm-1 (Fig. 2, A and B, red lines) (47Siebert F. Isr. J. Chem. 1995; 35: 309-323Crossref Scopus (82) Google Scholar, 48Siebert F. Mäntele W. Gerwert K. Eur. J. Biochem. 1983; 136: 119-127Crossref PubMed Scopus (98) Google Scholar). Subsequently, bands at 1748/68 cm-1, 1643 cm-1, and 1556 cm-1 reflect an increasing contribution of a Meta II minus rhodopsin difference spectrum. After a few seconds the final state is reached and the spectrum exhibits now the typical Meta II difference bands. The black line in Fig. 2B is the last spectrum recorded 8 s after the activating flash. The green lines in Fig. 3 show the kinetics of selected characteristic FTIR bands, representing the formation of active Meta II as extracted from a three-dimensional plot, recorded at 10 °C and pH 6 (spectral resolution 8 cm-1, time resolution 16.2 ms). Difference bands reflecting proton transfer processes, changes in hydrogen bonding, and the secondary structure occurring during the formation of Meta II show kinetics similar to those observed in the literature (49Parkes J.H. Gibson S.K. Liebman P.A. Biochemistry. 1999; 38: 6862-6878Crossref PubMed Scopus (31) Google Scholar) and to our own time-resolved UV-visible spectra shown in Fig. 1. This applies to the band at 1748 cm-1 (A) indicating changes of the hydrogen bonding environment of Asp83 and of Glu122, to the bands at 1643 cm-1 (B; amide I-region) and at 1556 cm-1 (C; amide II-region and the C=C stretch of the chromophore) typical for changes in the secondary structure of the protein and to the band at 1713 cm-1 (D) characteristic for the protonation of the Schiff base counterion Glu113. These bands appear with comparable kinetics as observed for the 380 nm absorption shown in the inset of Fig. 1 reflecting Schiff base deprotonation in Meta II. The band at 1205 cm-1 (E), a marker band of both the protonated Schiff base in Meta I and of the 9-cis-retinal in the isorhodopsin ground state shows a biphasic behavior: a fast increase in absorption (t½ < 5 ms) due to the formation of isorhodopsin and Meta I, followed by a slower decrease (t½ ≈ 90 ms) representing formation of Meta II from Meta I. The negative band at 1238 cm-1 (F, green line) in the chromophore fingerprint region is typical for the light-induced 11-cis to all-trans isomerization, a process already completed in the early photointermediates. Therefore, this event is too fast to be resolved with the FTIR rapid-scan technique. Deactivation—We return to Fig. 2, to see FTIR difference spectra of light-induced deactivation. In Fig. 2C, at pH 6 and 10 °C, 25 ms after a blue flash, a difference spectrum is observed with a characteristic, intense difference band at 1556 cm-1 (red line in Fig. 2, C and D). However, most other spectral regions display only minor difference bands. Subsequently, difference bands typical for Meta III, including the Meta III marker band at 1348 cm-1, arise within 8 s (Fig. 2D, black line). The blue lines in Fig. 3 show the kinetics of selected difference bands characteristic for the light-induced deactivation. Interestingly, the kinetics is different from the activating pathway. Bands assigned to deactivating changes of hydrogen bonding or carboxylic acids or to changes of the secondary structure of the receptor occur on a much slower time scale than observed during the activation. Generally, most of the bands representing the deactivation process show a biphasic behavior with an additional fast component (t½ < 15 ms), which cannot be resolved with the available setup. For deactivation, the half time of the band at 1748 cm-1 (A), showing changes of the carboxylic acid residues Glu122 and Asp83 is t½ = 1700 ms and of the band at 1643 cm-1 (B), assigned to the amide I vibration and thus sensitive to changes of the secondary structure, is t½ = 1350 ms. These kinetics are not only significantly slower than the kinetics of the reprotonation process of the Schiff base during the deactivation (see blue line in the inset of Fig. 1B and Fig. 3, D and E) but are even slower than the kinetics of the same bands during the activation (green lines in Fig. 3, A and B). For comparison, the time constants for the 1748 cm-1 band during activation is 150 ms (1700-ms deactivation) and 80 ms (1350-ms deactivation) for the 1643 cm-1 band (secondary structure). The intense band at 1556 cm-1 (C), arising within a few milliseconds after the blue flash, indicates the formation of a species, which we term RR-Meta. The difference spectrum of this intermediate is ide