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Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Activation and the Role of the Retinal
  5. Blue-Light Absorption by Meta II leads to Meta III and Not to Rhodopsin
  6. Light-Induced Deactivation and the Role of the Retinal
  7. Photointermediates of Meta III
  8. Physiological Implications of the RR-Meta/Meta III Equilibrium
  9. Concluding Remarks
  10. Acknowledgments
  11. References

Rhodopsin, the visual pigment of the rod photoreceptor cell contains as its light-sensitive cofactor 11-cis retinal, which is bound by a protonated Schiff base between its aldehyde group and the Lys296 side chain of the apoprotein. Light activation is achieved by 11-cis to all-trans isomerization and subsequent thermal relaxation into the active, G protein-binding metarhodopsin II state. Metarhodopsin II decays via two parallel pathways, which both involve hydrolysis of the Schiff base eventually to opsin and released all-trans retinal. Subsequently, rhodopsin's dark state is regenerated by a complicated retinal metabolism, termed the retinoid cycle. Unlike other retinal proteins, such as bacteriorhodopsin, this regeneration cycle cannot be short cut by light, because blue illumination of active metarhodopsin II does not lead back to the ground state but to the formation of largely inactive metarhodopsin III. In this review, mechanistic details of activating and deactivating pathways of rhodopsin, particularly concerning the roles of the retinal, are compared. Based on static and time-resolved UV/Vis and FTIR spectroscopic data, we discuss a model of the light-induced deactivation. We describe properties and photoreactions of metarhodopsin III and suggest potential roles of this intermediate for vision.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Activation and the Role of the Retinal
  5. Blue-Light Absorption by Meta II leads to Meta III and Not to Rhodopsin
  6. Light-Induced Deactivation and the Role of the Retinal
  7. Photointermediates of Meta III
  8. Physiological Implications of the RR-Meta/Meta III Equilibrium
  9. Concluding Remarks
  10. Acknowledgments
  11. References

Rhodopsin is the visual pigment in rod photoreceptor cells enabling vision under dim light conditions. It is composed of seven transmembrane spanning helices and an eighth helix running parallel to the cytoplasmic surface (1,2). The light-sensitive cofactor making rhodopsin a quantum detector is the vitamin A derivative 11-cis retinal. In the ground state, the retinal is fixed to the protein on the one hand by hydrophobic interactions between its beta ionone ring and amino acid side chains of the binding pocket, and on the other hand, by a protonated Schiff base linkage between the aldehyde group of 11-cis retinal and the side chain of Lys296 (3,4). The positive charge of the protonated Schiff base is neutralized and thereby stabilized by a salt bridge between Schiff base and its counterion Glu113 (5).

Activation of rhodopsin starts with light-induced isomerization of 11-cis retinal around the C11 = C12 double bond (6,7), leading to the early intermediate bathorhodopsin (Batho) that contains the chromophore in a strained all-trans configuration (8–10). Subsequently, the receptor relaxes thermally through the intermediates lumirhodopsin (Lumi) and metarhodopsin I (Meta I), each characterized by a specific UV/Vis absorption maximum and a typical infrared difference spectrum. The activation process depends on a proton transfer from the Schiff base to the counterion Glu113, which marks the transition to metarhodopsin IIa (Meta IIa) and on a spectroscopically silent proton uptake event, leading to the active, G protein-binding Meta IIb state (11). Meta II decays on a timescale of minutes mainly by hydrolysis of the Schiff base into opsin and all-trans retinal. The release of retinal from the binding site allows the receptor to adopt a largely inactive conformation (12–14). Apart from this direct decay, an alternative pathway of receptor deactivation is provided by the formation of metarhodopsin III (Meta III), which absorbs at 475 nm, and eventually decays into opsin and all-trans retinal, albeit on a timescale of hours (15–18).

A complicated and time consuming cellular machinery, termed retinoid cycle, converts released all-trans back to 11-cis retinal, which can then recombine with opsin to form rhodopsin's ground state. Dysfunctions of this cycle lead to severe eye diseases, e.g. macular degeneration such as Retinitis pigmentosa or Stargardt's disease (19).

Despite its drawbacks, the retinoid cycle seems to be the only efficient way to regenerate rhodopsin, as experimental attempts to restore the ground state by illumination of the active state have failed. Instead, recent work has shown that illumination of Meta II does not lead back to the ground state by trans/cis isomerization of the polyene chain, but to the formation of rather inactive Meta III by syn/anti isomerization of the Schiff base (20,21), the same species as observed as a side product of thermal Meta II decay (22,23). This is in remarkable contrast to archaeal and invertebrate rhodopsins where the ground state can be regenerated by light-induced mechanisms (24).

In this contribution, we summarize our current knowledge on the mechanism of light-induced Meta II deactivation leading to Meta III, mainly on the basis of results obtained by a combination of static and time-resolved UV/Vis and FTIR difference spectroscopy. Based on a comparison of activating and deactivating pathways and of the switching mechanisms, the roles of the retinal are discussed. In the face of its properties, we suggest potential roles of Meta III for the visual system.

Activation and the Role of the Retinal

  1. Top of page
  2. Abstract
  3. Introduction
  4. Activation and the Role of the Retinal
  5. Blue-Light Absorption by Meta II leads to Meta III and Not to Rhodopsin
  6. Light-Induced Deactivation and the Role of the Retinal
  7. Photointermediates of Meta III
  8. Physiological Implications of the RR-Meta/Meta III Equilibrium
  9. Concluding Remarks
  10. Acknowledgments
  11. References

Activation of rhodopsin is achieved by light-induced 11-cis to all-trans retinal isomerization around the C11 = C12 double bond and subsequent thermal relaxation into the active Meta II state, proceeding through a number of spectroscopically resolvable intermediates such as Batho, Lumi and Meta I (3,5). These activating steps are illustrated in Fig. 1.

image

Figure 1.  The activating pathway of rhodopsin. In rhodopsin’s dark state the chromophore 11-cis-15-anti retinal (red) is bound to the apoprotein by a protonated Schiff base, stabilized by its counterion Glu113 (yellow). Green light absorption (λ ≥ 500 nm) induces 11-cis-15-anti to all-trans-15-anti isomerization of the retinal. This isomerization (wavy arrow) triggers the formation of the early intermediate Batho, which relaxes thermally on a millisecond timescale through the intermediates Lumi and Meta I into the active state (solid arrows). A crucial step for activation is the breakage of the salt bridge by proton transfer from the the Schiff base to its counterion Glu113 during transition from Meta I to Meta IIa. For the formation of active form Meta IIb, proton uptake from the bulk phase, probably by Glu134 is required. Note that this proton uptake is enabled by the scaffold function of the retinal, so that it can even occur at unfavorable high pH of the bulk phase (forced protonation).

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Batho (λmax = 540 nm), the first intermediate of the activation pathway, stores two-thirds of the absorbed light energy and contains the retinal in a strained all-trans-15-anti configuration. The mechanism of subsequent thermal relaxation of this strain is under debate. In photo affinity labeling studies a flip-over of the beta ionone ring from Trp265 in helix VI to the Ala169 in helix IV during the formation of the Lumi intermediate was suggested. This step, in which the beta ionone ring leaves the binding pocket, could account for a partial relaxation of the strain (25).

A movement of the ring towards helices III and IV was also observed in a recent crystallographic study; however, a ring displacement outside the binding pocket was excluded (26). The flip mechanism was further challenged by electron microscopy (27) and by solid state NMR studies, which both indicated that the beta ionone ring largely remains in its original position upon formation of Meta I (28). Even during the transition to Meta II, only slight changes in the hydrophobic interactions between the ring and the binding pocket could be detected, which concern the methyl group at C17 and C18 (29). This was supported by FTIR studies which showed that the formation of the early intermediates is not affected when the beta ionone ring is truncated, as long as the methyl groups at C17 and C18 are present (30–32).

These small changes in the interactions between the ring and the binding pocket may pave the way for the formation of the active state, involving the crucial proton transfer from the Schiff base to its counterion Glu113. This event disrupts the salt bridge between these two groups, which is one of the factors that holds the receptor in its inactive conformation. This step is mainly responsible for the significant shift of the absorption maximum from 480 nm in Meta I to 380 nm in Meta IIa (22). However, a spectroscopic silent proton uptake, presumably by Glu134 or a hydrogen bonded network in which it is involved, is necessary for the formation of the active Meta IIb state, which finally binds and activates the G-protein (11,33). The intermediates Meta I and Meta II are in a conformational equilibrium that depends on pH and temperature (22,34,35) and other factors such as lipid environment (36) and pressure (37). It is noteworthy that this essential proton uptake occurs although under physiological conditions the pH of the bulk phase is higher than the intrinsic pKa of the proton uptake group (forced protonation) (38,39).

The immediate question arising in this context is how the chromophore interacts with the apoprotein to enforce this last protonation, which presumably triggers rhodopsin activation.

A possible answer comes from studies in which rhodopsin was regenerated with various retinal analogues. It was shown that rhodopsin containing 11-cis-9-demethyl-(9-dm-Rho) or 11-acyclic-retinal (ac-Rho) still establishes the equilibrium between an inactive Meta I like state and an active Meta II like state. However, under physiological pH the equilibrium is largely on the Meta I side and is only shifted significantly towards Meta II at low pH of the bulk phase (around pH 5), i.e. the intrinsic pKa of the proton uptake group (31,32,40,41).

Similar observations were made for pigments regenerated with ring-constrained 11-cis retinal analogues (ring-constrained-rho), preventing isomerization around the C11 = C12 double bond (42). These pigments can exist in cis- and trans- like isoforms, which undergo light-induced conversion into each other. The isomerization which does not affect the blocked C11 = C12 double bond, but two other double bonds in the 9 and 13 position of the polyene chain, does not lead to a fully elongated all-trans form and activates the receptor only marginally at physiological pH. However, when the pH of the bulk phase is low, the equilibrium between the inactive and the active form is significantly shifted towards the active conformation (43,44). In Fig. 2, we show the share of the active Meta II like form in the equilibrium as a function of pH for 9-dm-Rho (blue line), ac-Rho (red line) and ring-constrained-rho (green line). The curve for native rhodopsin (black line) is given for comparison.

image

Figure 2.  The Meta I/Meta II equilibrium. To investigate the role of the retinal during activation, we studied the effect of different retinal analogues on the equilibrium between inactive Meta I and active Meta II. The share of Meta II in the equilibrium of pigments regenerated with 9-demethyl-retinal, 11-cis-diethyl-acyclic-retinal and ring-constrained retinal and of wild-type rhodopsin is shown as function of pH at 20°C. The fraction was estimated by typical Meta II bands in the FTIR difference spectrum. The data were fitted by a hyperbolic function. Compared to native rhodopsin (pKa = 7.7, black line), pigments regenerated with modified retinals show a significant shift of the pKa towards lower values. The pKa for rhodopsin regenerated with ring constraint retinals (green) is 5.4, with 9-demethyl-retinal (blue) is 5.0 and with acyclic-retinal (red) is 5.2. This demonstrates that these pigments can adopt an active conformation only at low pH.

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In these studies, evidence was provided, that all-trans retinal acts as a rigid scaffold, which controls even the last step of the reaction, namely the essential proton uptake by Glu134. The function of this scaffold is to adjust donor and acceptor groups involved in this proton transfer reaction by affecting molecular angles, distances and electrical fields. This modulates the pKa of the intrinsic proton uptake group to finally achieve proton uptake from the bulk phase. This scaffold enables “forced protonation” as long as all structural determinants of the retinal, such as the 9-methyl group and an intact beta ionone ring are present and as long as the formation of the fully elongated all-trans form of the polyene chain is allowed.

This argues for a limited scaffold function of the retinal analogues, as the pKa of the equilibrium between the inactive and active conformations of these pigments resembles the pKa of the equilibrium between inactive and active conformations of the opsin apoprotein, which does not have any restrictions imposed by a bound chromophore (14). In this case, the active conformation can only be formed when proton uptake is favored by the surrounding pH.

Blue-Light Absorption by Meta II leads to Meta III and Not to Rhodopsin

  1. Top of page
  2. Abstract
  3. Introduction
  4. Activation and the Role of the Retinal
  5. Blue-Light Absorption by Meta II leads to Meta III and Not to Rhodopsin
  6. Light-Induced Deactivation and the Role of the Retinal
  7. Photointermediates of Meta III
  8. Physiological Implications of the RR-Meta/Meta III Equilibrium
  9. Concluding Remarks
  10. Acknowledgments
  11. References

UV/ Vis spectroscopy has been used by several groups to study light-induced deactivation of Meta II (22,45–47). We performed similar experiments with membrane bound rhodopsin. The data (Fig. 3) showed that light induced depletion of the active Meta II state (green line), absorbing at 380 nm, leads to a product with an absorption maximum around 475 nm (blue line). This species was identified as an inactive intermediate with a reprotonated Schiff base (48). It was speculated that this product might contain the chromophore as a mixture of several retinal isoforms, such as 11-cis, 9-cis and 7-cis. However, UV/Vis spectroscopy mainly reports about the protonation state of the Schiff base and thus the true nature of this product remained rather obscure. In a key experiment, we applied FTIR difference spectroscopy, as this technique is sensitive to changes in the apoprotein conformation and of the chromophore configuration.

image

Figure 3.  UV/Vis spectroscopy of light-induced Meta II deactivation. The ground state with a protonated Schiff base (black) shows an absorption maximum around 500 nm. At pH 6, 20°C, illumination with green light (λ ≥ 500nm) induces formation of the active Meta II state indicated by a blueshifted absorption maximum at 380 nm (green), caused by Schiff base deprotonation. Blue illumination (λ ≈ 400 nm) of Meta II leads to formation of largely inactive Meta III containing a reprotonated Schiff base (λmax = 475 nm, blue). Black arrows indicate the wavelength of illumination.

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In Fig. 4a (upper dark green spectrum), we show the Meta II minus rhodopsin FTIR difference spectrum for the transition from rhodopsin to Meta II, where negative bands arise as a result of the ground state and positive bands are assigned to the active Meta II state. The negative band at 1238 cm−1 is caused by changes in the retinal geometry and by changes in protein/chromophore interactions linked to 11-cis to all-trans isomerization triggering the activation process. Typical for Meta II formation are the bands at 1748/1768 cm−1, which reflect changes in hydrogen bonding of Glu122 and Asp83. In addition, the band at 1713 cm−1 is indicative for the protonation of the counterion Glu113, and the band at 1644 cm−1 arises as a result of changes in the secondary structure of the peptide backbone (49,50).

image

Figure 4.  FTIR-difference spectroscopy and scheme of light-induced rhodopsin activation and deactivation. (a) Upper dark green spectrum: Meta II minus rhodopsin difference spectrum of the transition from rhodopsin to Meta II. Negative bands correspond to absorptions in the ground state while positive bands correspond to the active state. The spectral pattern in the region between 1800 and 1500 cm−1 reports about proton transfer reactions, changing in hydrogen bonding and structural changes linked to the formation of the active Meta II state. The intense negative band at 1238 cm−1 is indicative for the 11-cis- to all-trans-isomerization of the retinal polyene chain. Blue: Meta III minus Meta II difference spectrum of blue–light-induced Meta II deactivation. The spectrum is a mirror image of the Meta II minus rhodopsin spectrum (dark green line) except in the chromophore fingerprint region between 1350 and 1100 cm−1. This shows that activating structural changes are all reverted, but the trigger mechanism is different. Instead of the band at 1238 cm−1, bands at 1348 cm−1 and at 1181 cm−1 appear, indicating Schiff base syn/anti isomerization. Light green: Reactivation of Meta III: Meta II minus Meta III difference spectrum obtained by illumination of Meta III with green (λ ≈ 470 nm) light. This spectrum and the spectrum of Meta II-deactivation (blue) are a perfect mirror image of each other including the chromophore fingerprint region (gray shaded). The scheme in Fig. 4b illustrates light-induced and thermal conversions between the different rhodopsin intermediates. Illumination of the ground state induces 11-cis-15-anti to all-trans-15-anti isomerization (first switch) and leads via several intermediates to Meta II (dark green). Blue illumination of Meta II triggers the formation of Meta III by anti/syn isomerization of the Schiff base C = N double bond (blue; second switch). Meta III is still light-sensitive and can be reconverted to Meta II by green light (light green line), starting with syn/anti isomerization. Consequently, the second switch is reversible and operates a cycle between Meta II and Meta III, which is also formed as a side product of thermal Meta II decay (black line). Thus, regeneration of rhodopsin′s dark state necessitates the metabolic retinoid cycle located in the pigment epithelium (RPE) (dashed gray line).

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The spectrum of the light-induced deactivation of Meta II is shown in Fig. 4a as a blue line. In this spectrum, bands assigned to structural changes in the apoprotein, which previously have occurred during receptor activation, are reverted. This affects particularly the amide bands at 1644 and 1555 cm−1 and the carboxylic acid region between 1800 and 1700 cm−1. In these regions, the spectrum is inverse to the spectrum of the activation process, showing that the receptor adopts an inactive conformation.

This, however, does not apply for the spectral region indicative for retinal geometry (gray shaded area in Fig. 4a, upper spectra). The band at 1238 cm−1, characteristic for the 11-cis/all-trans isomerization of the forward pathway is replaced by a different pattern around 1181 cm−1 and an additional band at 1348 cm−1 (20). It was later shown by FTIR spectroscopy with labeled retinals and by comparison of the spectrum with the thermal Meta II decay product Meta III, that the blue-light photoproduct is identical with Meta III that contains all-trans-15-syn retinal in its binding pocket (21). Therefore, a syn/anti isomerization of the C=N double bond of the Schiff base initiates the conformational changes of blue-light deactivation.

Meta III is still light-sensitive and can be converted back to Meta II by absorption of green light (Fig. 4, lower light green spectrum). Therefore, the syn/anti isomerization involved in the conversion of Meta III to Meta II is reversible as indicated by the reversed bands in the chromophore region (Fig. 4, blue line, gray shaded area). This is in contrast to the cis/trans isomerization which triggers activation starting from rhodopsin. To distinguish between these two trigger mechanisms we introduced the concept of the first and second switch: the first switch operates the transition from rhodopsin to Meta II and the second switch operates the transition from Meta II to Meta III (21). In Fig. 4b, the photoreactions of rhodopsin, Meta II and Meta III are summarized in a schematic view.

From these findings, it is obvious that the retinoid cycle is the only way to regenerate rhodopsin efficiently, as Meta III, the photoproduct of blue-light absorption, contains the chromophore in the all-trans-15-syn configuration instead of 11-cis as rhodopsin, resulting in different properties as described in detail below.

FTIR and UV/Vis measurements showed that Meta III is not as stable as rhodopsin, but decays into opsin and all-trans retinal with a half time of about 2 h at 30°C (17). Moreover, in the presence of G-protein or arrestin the Meta III decay is greatly accelerated and its half time is in the range of minutes (21,51–53). This suggests that Meta III shows a certain activity towards the G-protein and arrestin in contrast to rhodopsin where the specific interactions between the 11-cis retinal and the binding pocket protect the receptor against spontaneous thermal activation and depletion by signaling proteins, making it suitable as a single quantum light detector.

Light-Induced Deactivation and the Role of the Retinal

  1. Top of page
  2. Abstract
  3. Introduction
  4. Activation and the Role of the Retinal
  5. Blue-Light Absorption by Meta II leads to Meta III and Not to Rhodopsin
  6. Light-Induced Deactivation and the Role of the Retinal
  7. Photointermediates of Meta III
  8. Physiological Implications of the RR-Meta/Meta III Equilibrium
  9. Concluding Remarks
  10. Acknowledgments
  11. References

The combination of static UV/Vis and FTIR measurements contributed considerably to our current knowledge of the structural and functional properties of Meta III and about its reactivation to Meta II. However, these techniques are insufficient to provide information about the light-induced deactivation pathway from Meta II to Meta III; because of the shift in the Meta I/Meta II equilibrium towards Meta I at low temperatures, it is impossible to stabilize temperature trapped intermediates of blue–light-induced deactivation. To overcome this problem, a combination of time-resolved UV/Vis and time-resolved FTIR spectroscopy was applied for the first time to explore the deactivation pathway and to gain information about its kinetics and intermediate states (54).

In a first attempt, rhodopsin was activated by a green flash and subsequent UV/Vis spectra were taken every millisecond at 10°C. In Fig. 5a, green line, the change of the 380 nm absorption after a green flash, as a result of the deprotonation of the Schiff base is shown. The half time of this process was calculated to be 70 ms by a single exponential fit to the time course. A blue, deactivating flash was then applied to Meta II and the decrease in the 380 nm absorption (Fig. 5a, blue line) indicating the reprotonation of the Schiff base, was recorded. Surprisingly, the kinetics of this band is faster than 5 ms and could not be fully resolved with the available setup. This experiment clearly showed that the reprotonation step of the Schiff base during deactivation is much faster than the deprotonation of the Schiff base during activation. At first glance, this surprising result might suggest that the light-induced deactivation process is in general faster than activation. However, as mentioned above, UV/Vis spectroscopy mainly provides information about the protonation state of the Schiff base. Hence, to obtain the kinetics of conformational changes of the apoprotein, time-resolved FTIR difference spectroscopy was applied. Spectra were taken every 25 ms after the green or blue flash. In Fig. 5b and c, we show the kinetics of the band at 1713 cm−1, assigned to the protonation or deprotonation of the counterion Glu113, and of the band at 1644 cm−1 which is a general marker for changes of the secondary structure. The green lines correspond to the activation and the blue lines to the deactivation pathway.

image

Figure 5.  Time-resolved UV/Vis and FTIR difference spectroscopy of rhodopsin activation and deactivation. (a) Green line: Time-dependent change in the 380 nm absorption after activation of rhodopsin by a green flash (λmax ≈ 520 nm). Blue line: Time-dependent change in the 380 nm absorption after deactivation of rhodopsin by a blue flash (λmax ≈ 400 nm). Exponential fits to the data yield half times of 70 ms and <5 ms, respectively. (b and c) Green lines: Time course of the FTIR difference bands at 1713 cm−1 and 1644 cm−1 indicative for protonation of the counterion and changes of the secondary structure after flash activation of rhodopsin. Blue lines: Time course of the bands at 1713 and 1644 cm−1 following flash induced deactivation of Meta II. All experiments shown here were performed at 10°C, pH 6. Deprotonation of the Schiff base, protonation of the counterion and changes of secondary structure are tightly coupled during activation as seen by the similar kinetics. The coupling is lost during deactivation as indicated by the slow change of the band at 1644 cm−1, indicative for secondary structure. (d) Upper trace: Blue-light photoproduct minus Meta II FTIR difference spectrum recorded 25 ms after the deactivating flash, representing the RR-Meta intermediate. It is dominated by one large positive band in the amide II region at 1555 cm−1, while other bands occur only to a minor extent, arguing for a structural similarity of the photoproduct RR-Meta to Meta II. Middle trace: Photoproduct minus Meta II difference spectrum, recorded at pH 4.5. The spectrum was taken 20 s after illumination and also shows the formation of RR-Meta. Lower trace: RR-Meta minus Meta II difference spectrum recorded under conditions favoring the formation of Meta III (pH 7.0), in the presence of a G protein C-terminal peptide 8 s after light-induced deactivation. The formation of Meta III is blocked showing an activity of RR-Meta towards the peptide.

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The green traces in Fig. 5 show that for the activation pathway the kinetics of Schiff base deprotonation (a), counterion protonation (b) and structural changes of the apoprotein (c) occur with comparable kinetics (t1/2 = 70–100 ms), indicating a tight coupling between Schiff base deprotonation and structural changes. The situation is completely different for the deactivation pathway (Fig. 5a–c, blue lines). Here, the Schiff base reprotonation (a) and the deprotonation of the counterion (b) also occur with comparable fast kinetics (t1/2 < 15 ms), which are faster than the respective process during activation. However, structural changes (c) leading to largely inactive Meta III occur on a much slower timescale (t1/2 = 1.4 s). This shows that the coupling between Schiff base proton transfer and subsequent structural changes of the apoprotein is lost. More detailed information about the deactivation process can be derived when considering the FTIR difference spectra given in Fig. 5d (54).

The first difference spectrum, recorded 25 ms after the deactivating flash, is dominated by a band at 1555 cm−1, whereas difference bands in other spectral regions occur only to a minor extent (Fig. 5d, upper spectrum). This indicates that although the Schiff base is already reprotonated as seen from UV/Vis the receptor is still in a Meta II like conformation. Interestingly, this intermediate which we termed RR-Meta can also be stabilized at low pH as seen from Fig. 5d, middle trace, or in the presence of a G protein-derived peptide (Fig. 5d, lower trace). These data suggest a different role of the retinal in deactivation and activation process. As already mentioned, during receptor activation the retinal acts as a rigid scaffold, thereby adjusting donor and acceptor groups to enable necessary proton transfer processes including the essential proton uptake even at high pH of the bulk phase (forced protonation). Thus light energy is partly used to enforce the last protonation step by shifting the pKa of the proton uptake group to the neutral range.

During deactivation, anti/syn isomerization leads to a breakdown of the retinal scaffold function. Light energy is merely used for fast reorientation of counterion and Schiff base allowing its fast reprotonation. It does not enforce the final proton release into the bulk phase, since the last step of deactivation that leads to Meta III can occur only at favorable, high pH. The stabilization of RR-Meta in the equilibrium between RR-Meta and Meta III in the presence of the peptide clearly shows that RR-Meta is an active species in equilibrium with a largely inactive form (Meta III). In contrast to 11-cis-15-anti retinal as in rhodopsin, all-trans-15-syn retinal in Meta III can neither stabilize a complete inactive intermediate nor can it maintain the active form. Therefore, it may function as a partial agonist.

Based on the time-resolved spectroscopic data, a model as shown in Fig. 6 was deduced. It starts from Meta II containing the chromophore in the all-trans-15-anti conformation. Light absorption induces isomerization to all-trans-15-syn leading to a first hypothetical intermediate which we term R-Meta (reverted Meta). In the next step, the reprotonation of the Schiff base marks the transition to RR-Meta, which occurs within milliseconds. Although this intermediate contains the chromophore in the all-trans-15-syn conformation with a protonated Schiff base as it is in Meta III, it is still active, as shown by its structural similarity to Meta II and by its high affinity towards a G protein-derived peptide (Fig. 5d). In the last step of the deactivating pathway proton release occurs into the bulk phase, which only occurs at high pH values (pKa ≈ 5 (54)) on a timescale of seconds, reflecting that all-trans-15-syn retinal is not able to keep the receptor in a completely inactive conformation as 11-cis does in the ground state.

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Figure 6.  The model of light-induced Meta II deactivation. Light absorption in Meta II (blue wavy arrow) induces all-trans-15-anti to all-trans-15-syn isomerization of the chromophore leading to the first, hypothetical photoproduct R-Meta which contains a deprotonated Schiff base as in Meta II. The next step, the reprotonation of the Schiff base, marks the transition to the spectroscopic resolvable RR-Meta (λmax = 463 nm). It is formed on a millisecond timescale and is still structurally and functionally similar to Meta II. Deactivating structural changes of the protein leading to Meta III occur subsequently on a timescale of seconds (solid arrows) and only when a proton release to the bulk phase is favored by alkaline pH, which argues for a loss of the retinal’s scaffold function during deactivation (cf. Fig. 1).

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Photointermediates of Meta III

  1. Top of page
  2. Abstract
  3. Introduction
  4. Activation and the Role of the Retinal
  5. Blue-Light Absorption by Meta II leads to Meta III and Not to Rhodopsin
  6. Light-Induced Deactivation and the Role of the Retinal
  7. Photointermediates of Meta III
  8. Physiological Implications of the RR-Meta/Meta III Equilibrium
  9. Concluding Remarks
  10. Acknowledgments
  11. References

As noted above, Meta III containing retinal in the all-trans-15-syn configuration is a light-sensitive pigment (17,20,22). Its photochemistry is rather complex, as light absorption triggers two different pathways which occur in parallel, as observed by time-resolved FTIR and flash photolysis experiments (55,56).

On the one hand, when Meta III is activated by a short, green laser flash, one observes Schiff base syn/anti isomerization to all-trans-15-anti retinal, thus forming an early photointermediate which relaxes thermally through several intermediates into the Meta I/ Meta II equilibrium. These intermediates will be discussed later.

On the other hand, in parallel to syn/anti isomerization of the Schiff base, retinal isomerization around the polyene chain occurs, leading to either 11-cis-15-syn or 9-cis-15-syn retinal. These configurations are rather instable and decay on a timescale of seconds via a subsequent thermal syn/anti isomerization to the rhodopsin or to the isorhodopsin ground state.

However, under conditions of continuous illumination, absorption of a second photon can occur, and rhodopsin and isorhodopsin are converted to Meta II so that eventually the formation of this species is observed (cf. Fig. 7, wavy arrows reflect the absorption of one photon at the respective wavelength).

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Figure 7.  The light-induced activation of Meta III. Light-sensitive Meta III, absorbing at 475 nm, contains the chromophore in the all-trans-15-syn configuration. Absorption of light triggers two parallel pathways. Wavy arrows indicate light induced, solid arrows thermal transitions. On the one hand, Schiff base syn/anti isomerization leads to the M-Batho photoproduct which can be trapped at 80 K. This reaction is photoreversible, since M-Batho can be reconverted to Meta III. At appropriate temperatures the protein relaxes through the M-Lumi intermediate into the pH- and temperature-dependent equilibrium between Meta I and Meta II. On the other hand, a parallel pathway starts with trans/cis isomerization of the retinal. It leads to thermally instable photoproducts with a 9-cis-15-syn or 11-cis-15-syn chromophore. These species decay by a thermal Schiff base syn/anti isomerization to the ground state (9-cis-15-anti or 11-cis-15-anti) on a timescale of seconds. Absorption of a second photon as occurring under conditions of continuous illumination, activates the usual pathway via Batho, Lumi and Meta I that ends up in Meta II.

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At low temperatures trans/cis isomerization is not observed anymore in the binding pocket of Meta III. Instead, syn/anti isomerization of the Schiff base becomes the predominant photoreaction (56). In analogy to rhodopsin activation, this allowed us to trap early photointermediates of Meta III at 80 and 173 K (21), which we term M-Batho and M-Lumi, respectively. The photoproduct minus Meta III FTIR difference spectra of these intermediates are very similar to the corresponding spectra of rhodopsin activation. Differences are apparent in those spectral regions indicative for the chromophore geometry (data not shown), showing that all-trans-15-syn/all-trans-15-anti isomerization triggers the formation of M-Batho and 11-cis-15-anti/all-trans-15-anti isomerization the formation of Batho. Hence, the retinal geometry in M-Batho is the same as in Batho, arguing for a shared identity between these two intermediates.

For further characterization, both intermediates were probed by light, and the respective photoproducts were compared. In Fig. 8a, we show the difference spectrum of a photostationary mixture of rhodopsin and isorhodopsin illuminated with green light at 80 K (Batho conditions) as the blue line, whereas the green line represents the difference spectrum of the photoreversal induced by illumination with orange light (λmax > 540 nm). The spectra are inverses of one another, demonstrating that the reaction is perfectly reversible. Consequently, as seen by the bands 1238 and 1206 cm−1, illumination of Batho leads back to the formation of rhodopsin and isorhodopsin.

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Figure 8.  Infrared difference spectroscopy of the early photointermediates of rhodopsin/isorhodopsin and Meta III. Rhodopsin and Meta III were exposed to green light at 80 K to produce the Batho and M-Batho intermediates. (a) Blue line: Batho minus dark state difference spectrum starting from a photostationary mixture of rhodopsin and isorhodopsin. Green line: Difference spectrum obtained after illumination of Batho with orange light (λ > 520 nm) at 80 K. The spectra are inverses of one another and the bands at 1206 and 1238 cm−1 show the formation of the rhodopsin and isorhodopsin by isomerization of the polyene chain around the C11 = C12 and C9 = C10 double bonds, respectively. (b) Similar experiment as in Fig. 8a, starting from Meta III. Blue line: Difference spectrum M-Batho minus Meta III at 80 K. The bands at 1181 and 1348 cm−1 indicate Schiff base isomerization from all-trans-15-syn to all-trans-15-anti. Green line: Difference spectrum of the photoreversal recorded after illumination with orange light. Analogue to the spectra in (a) these spectra are inverses of one another, including the chromophore fingerprint. Hence, different switches operate in Batho and M-Batho. Illumination of M-Batho leads to the formation of Meta III, while illumination of Batho leads to the formation of rhodopsin and isorhodopsin. This suggests that these two intermediates are not identical.

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The same experiment was performed starting from Meta III (Fig. 8b, blue line, M-Batho minus Meta III, green line photoreversal). These spectra are also inverses of one another, and the bands at 1181 cm−1 and at 1348 cm−1, occurring in both spectra, demonstrate that illumination of M-Batho leads to the formation of Meta III and not to the formation of rhodopsin. We conclude that although Batho and M-Batho bear the chromophore in the all-trans-15-anti configuration, these intermediates are not identical. Different protein–chromophore interactions in these two intermediates allow Schiff base anti/syn isomerization in M-Batho and trans/cis isomerization in Batho, directing the photoreactions into different products. Similar observations were made for M-Lumi and Lumi (21) (data not shown).

It is interesting to note that the illumination of M-Meta I does not lead to the formation of Meta III as observed for the earlier intermediates M-Batho and M-Lumi, but to the formation of rhodopsin and isorhodopsin. This means that the photoreaction flows into a common Meta I intermediate regardless of whether it starts from Meta III or rhodopsin, demonstrating that the activating pathway of Meta III culminates in the usual Meta I/Meta II equilibrium (21). The photoreactions and intermediates of Meta III are illustrated in Fig. 7.

Although the specific reasons for this peculiar behavior are still unknown, it is obvious that there are alterations in the binding pocket during the transition from M-Lumi to Meta I that do not occur during the Lumi to Meta I transition. These changes might account for a change in selectivity from anti/syn to cis/trans isomerization. The situation is even more complicated by the fact that during the transition from Lumi to Meta I, where no change of the isomerization preference is observed, significant changes of the immediate environment of the chromophore were detected by low temperature FTIR spectroscopy (57).

Another change in the isomerization specificity is observed during the transition from Meta I to Meta II, since Meta I still allows photoreversal back to the ground state, whereas in Meta II syn/anti isomerization of the Schiff base leading to Meta III occurs (21).

Key events which might account for this change in specificity during the formation of Meta II are probably conformational changes of the receptor forcing the retinal into a more planar geometry (58) and the deprotonation of the Schiff base changing the electronic structure of the polyene chain (18). The deprotonation of the Schiff base, however, cannot be the only reason, since M-Batho and M-Lumi, both with a protonated Schiff base, undergo syn/anti isomerization triggering formation of Meta III.

Physiological Implications of the RR-Meta/Meta III Equilibrium

  1. Top of page
  2. Abstract
  3. Introduction
  4. Activation and the Role of the Retinal
  5. Blue-Light Absorption by Meta II leads to Meta III and Not to Rhodopsin
  6. Light-Induced Deactivation and the Role of the Retinal
  7. Photointermediates of Meta III
  8. Physiological Implications of the RR-Meta/Meta III Equilibrium
  9. Concluding Remarks
  10. Acknowledgments
  11. References

The finding that blue–light-induced deactivation of rhodopsin's active Meta II state does not efficiently lead back to the ground state by trans/cis isomerization of the polyene chain (first switch), but rather to RR-Meta/Meta III by anti/syn isomerization of the Schiff base (second switch), raises the question about the relevance of these intermediates for the vision process.

Under physiological pH and temperature, Meta III is relatively stable and decays on a timescale of hours. Since it contains the chromophore in the all-trans-15-syn configuration, Meta III could function as a storage form of all-trans retinal. The physiological benefit would be a temporary removal of all-trans retinal from the biochemical pathways and a decrease in the amount of free or membrane bound all-trans retinal (54). This could avoid the formation of deleterious side products because of an excess of all-trans retinal, which may cause eye diseases such as age related macular degeneration (59,60).

Beside pH and temperature, the depletion rate of Meta III strongly depends on the presence of signaling proteins such as transducin and arrestin (51–53). Although Meta III is stable for hours, its depletion rate is increased to the timescale of minutes in the presence of G protein-derived peptides. With these interactions, the Meta III storage photoproduct could be emptied and all-trans retinal could then flow into the regeneration cycle in a controlled manner. Therefore, the retinal release from Meta III could be regulated by different concentrations of transducin and arrestin, and thus possible regulatory mechanisms could be physiologically relevant.

Under the artificial conditions of extended blue illumination, up to 80% of the photoreceptor can be converted to Meta III, and it can be anticipated that a considerable amount of Meta III is also formed in the photoreceptor cell under native conditions. The relative stability of Meta III might be physiologically advantageous with respect to its function as a storage form for all-trans retinal, but it could be deleterious during dark adaptation, since the residual activity of Meta III and its equilibrium with active RR-Meta might increase the noise level in the photoreceptor cell (54,61). Thus, Meta III must probably be removed when conditions change from bright light to dim light. A faster and/or a more regulated depletion of the Meta III pool than achieved by thermal decay would therefore be desirable for a faster reduction of the noise level. An accelerated depletion of Meta III can possibly be induced by the interaction of transducin or arrestin with active RR-Meta, thereby shifting the equilibrium towards the faster decaying RR-Meta.

Alternatively, the visual system could benefit from the presence of Meta III, since the formation of Meta III reduces the amount of Meta II which has to be degraded to achieve dark adapation. Due to its light sensitivity, Meta III can act as a “noisy” but immediately available photoreceptor before the cell has fully dark adapted. However, the elucidation of this possible role of Meta III as a photoreceptor is challenging and requires more detailed analysis of Meta III in intact retinae or in vivo, particularly the determination of the exact amount of Meta III under physiological conditions.

Finally, we come back to our initial question why such a complicated biochemical machinery like the retinoid cycle is used to regenerate 11-cis from all-trans retinal, a step which in principle could be achieved by a simple photoreaction.

As we have seen, blue–light-induced deactivation of rhodopsin's active Meta II state leads to Meta III instead of rhodopsin. A thermal back-isomerization to the ground state as observed in related retinal proteins, such as the proton pump bacteriorhodopsin, is not observed either, since Meta II decays on the timescale of minutes (depending on pH conditions and/or the presence of G protein or arrestin) via two parallel pathways. Both pathways involve hydrolysis of the Schiff eventually leading into opsin and free all-trans retinal. We have shown that Meta III has a residual activity towards the G protein, which might cause a certain level of dark noise.

Evidence suggests that only the presence of 11-cis or 9-cis retinal in the binding pocket can protect the receptor efficiently against spontaneous activation by its high enthalpic barrier. Since 11-cis or 9-cis retinal is formed neither thermally nor in a light-induced way, the retinoid cycle provides the only possibility to efficiently restore rhodopsin, which can function as single quantum detector. One might speculate that the possibility of thermal or light-induced return of the all-trans retinal into 11-cis retinal as in the ground state would require a lower enthalpic barrier between the cis- and the trans forms. This in turn would then contribute to an increase in the level of spontaneous activation and therefore increase dark noise. Thus, the complicated retinoid cycle might be the high price the cell must pay for keeping rhodopsin dark-activity so low and consequently photoreceptor cell sensitivity so high.

Concluding Remarks

  1. Top of page
  2. Abstract
  3. Introduction
  4. Activation and the Role of the Retinal
  5. Blue-Light Absorption by Meta II leads to Meta III and Not to Rhodopsin
  6. Light-Induced Deactivation and the Role of the Retinal
  7. Photointermediates of Meta III
  8. Physiological Implications of the RR-Meta/Meta III Equilibrium
  9. Concluding Remarks
  10. Acknowledgments
  11. References

In recent years, improved spectroscopic methods have substantially contributed to our understanding of the photoreactions of vertebrate rhodopsin and of its photoproducts. Particularly the question whether the complicated and time-consuming retinoid cycle, which regenerates 11-cis retinal, can be shortcut by light was in the focus of our research. It is now clear that light-induced conversion of the active Meta II state does not lead back to the ground state but rather to the formation of light-sensitive Meta III, an intermediate with different properties as summarized in this review. In the photoreactions of rhodopsin, Meta II and Meta III, two different switching mechanisms were identified: The first switch is the cis/trans isomerization of the chromophore polyene chain, which triggers the activation of rhodopsin. The second switch triggers light-induced and thermal formation of Meta III and was identified as an anti/syn isomerization of the Schiff base C=N double bond. All-trans-15-syn retinal neither provides a scaffold that stabilizes the active form nor enables it the specific interactions necessary for complete inactivation of the receptor. Hence, other factors such as pH determine the activity of the protein, permitting an equilibrium between active RR-Meta and residual active Meta III.

This may result in a noise level orders of magnitude higher than in the dark ground state. The retinoid cycle, with its possible dysfunctions leading to severe eye diseases, is probably the price that has to be paid for such an exceedingly low noise ground state, since light absorption of the retinal in the active state does not enforce full deactivation.

Meta III can be reversibly converted to Meta II by light absorption. This opens the possibility to capture all-trans retinal within a light driven cycle and thus to keep it away from biochemical pathways in order to avoid the formation of toxic side products. A deeper understanding of the physiological role of Meta III is, however, still pending and depends on the development of more sophisticated techniques that allow investigations in vivo.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Introduction
  4. Activation and the Role of the Retinal
  5. Blue-Light Absorption by Meta II leads to Meta III and Not to Rhodopsin
  6. Light-Induced Deactivation and the Role of the Retinal
  7. Photointermediates of Meta III
  8. Physiological Implications of the RR-Meta/Meta III Equilibrium
  9. Concluding Remarks
  10. Acknowledgments
  11. References

Acknowledgements— We are grateful to K. P. Hofmann and Martin Heck for their contributions and helpful discussions. We want to thank Martha Sommer, Kerstin Zimmermann and Berhard Knierim for discussions and critical reading of the manuscript. The authors were generously supported by the Deutsche Forschungs Gemeinschaft (SfB 498 to F. J. Bartl).

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Activation and the Role of the Retinal
  5. Blue-Light Absorption by Meta II leads to Meta III and Not to Rhodopsin
  6. Light-Induced Deactivation and the Role of the Retinal
  7. Photointermediates of Meta III
  8. Physiological Implications of the RR-Meta/Meta III Equilibrium
  9. Concluding Remarks
  10. Acknowledgments
  11. References