Photoisomerization in rhodopsins: Shape-changing reactions of retinal at low temperatures Open Access

Rhodopsins, present in both animals and microbes, are a family of photoreceptive membrane proteins covalently bound with a retinal chromophore. Animal rhodopsin was first isolated from animal retina in 1876 as a visual pigment in animal retina.¹ Almost a century later, the first microbial rhodopsin was found in a halophilic archaea as a light-driven proton-pump bacteriorhodopsin (BR) from Halobacterium salinarum.² Animal rhodopsins are G protein–coupled receptors,^3–7 whereas microbial rhodopsins have widely variable functions and can act as light sensors, light-gated ion channels, light-driven ion pumps, and light-activated enzymes.^7–11 In view of their functional diversity and newly emerging mutations, microbial rhodopsins have emerged as important tool proteins in optogenetic research and have helped revolutionize neurobiology and brain science.^12–14 

Animal and microbial rhodopsins, respectively, bind to 11-cis and all-trans retinal via a Schiff base linkage with a lysine residue in the seventh helix [Figs. 1(a)–1(c)],^3–11 while a negatively charged counterion stabilizes the protonated Schiff base [Figs. 1(d) and 1(e)]. The chromophore absorbs light triggering a primary photoreaction (11-cis to all-trans and all-trans to 13-cis retinal isomerizations in animal and microbial rhodopsins, respectively; Fig. 2) and subsequent protein conformation changes accounting for each function. Given that isomerization involves changes in the molecular structure, the process cannot occur if the solvent is frozen at temperatures lower than the melting point of the solvent. Therefore, the mechanism of the currently proposed photochemistry of rhodopsin remained debatable, as the above isomerizations occur even at cryogenic temperatures (e.g., 77 K).^15,16

The isomerization hypothesis has been thoroughly investigated. In particular, the primary photoreaction was first probed by low-temperature spectroscopy, which enabled the stabilization of primary photo-intermediates at low temperatures. In the 1970s, the photochemistry of bovine rhodopsin was examined using the newly emerged picosecond pulsed laser methods. Synthetic retinal analogs with locked 11-cis (for animal rhodopsin) and 13-trans (for microbial rhodopsin) conformations play key roles in identifying retinal chromophore isomerization as the primary photoreaction. In the 1990s, femtosecond laser spectroscopy revealed that the chemical reaction occurring in rhodopsins is one of the fastest known to date. Thus, the potential barrier along the isomerization coordinate in the excited state is very low, which explains the occurrence of the shape-changing reaction at cryogenic temperatures. Such low-barrier isomerization also well explains the efficiency and high quantum yields of the photoreactions (>0.5) typically observed in animal and microbial rhodopsins.

This review summarizes the photoreactions occurring in rhodopsins, focusing on the debatable isomerization hypothesis. We start by describing early studies on photoreactions in rhodopsins, highlighting the fact that works on animal rhodopsins preceded those on microbial rhodopsins. Initially, low-temperature spectroscopy was the main investigation method, while the picosecond laser photolysis of proteins containing synthetic 11-cis- or all-trans-locked retinal was used to test whether isomerization is the first reaction or not. Later, femtosecond laser pulse techniques directly captured the electronically excited states of rhodopsins undergoing isomerization. The ultrafast rate of isomerization suggests that the protein environment lacks the time to change its structure, and the occurrence of shape-changing reactions without large structural changes was indeed confirmed by structural biology methods, namely, vibrational spectroscopy and x-ray crystallography analyses of primary intermediates. Thus, the photoisomerization of rhodopsins at low temperatures is well established. However, near-infrared-absorbing microbial rhodopsins have been shown to exhibit exceptional behavior as they do not undergo low-temperature photoreactions.

Since the discovery of animal rhodopsins, they have been exclusively used in early-stage photoreaction research. In the 1930s, George Wald discovered that retinal is a rhodopsin chromophore. Tôru Yoshizawa and colleagues used low-temperature spectroscopy to detect a red-shifted photoproduct generated from bovine rhodopsin (prelumirhodopsin, currently known as bathorhodopsin) at 87 K, revealing that it was converted back to rhodopsin upon illumination¹⁵ and, once warmed to >123 K, reverted to lumirhodopsin and degraded to all-trans retinal and opsin via a number of intermediates. The results of low-temperature spectrophotometric investigations revealed that bathorhodopsin possesses a highly stiff and distorted all-trans retinal chromophore and features a higher-lying energy state than rhodopsin and the subsequently formed intermediates.¹⁶ 

Yoshizawa et al. proposed the cis–trans isomerization of the retinal chromophore to be the primary process responsible for the transformation of rhodopsin to bathorhodopsin. However, the cis–trans isomerization is a shape-changing reaction that involves non-superimposable 11-cis and all-trans chromophores [Fig. 2(a)]. The isomerization hypothesis suggests the formation of a highly constrained and distorted structure. However, the primary reaction responsible for animal vision in the context of rhodopsins remains unanswered. In fact, the results of the picosecond laser photolysis monitoring highlight one of the problems of the isomerization hypothesis. Specifically, bathorhodopsin formation occurs within 6 ps after rhodopsin excitation at room temperature,¹⁷ i.e., is overly fast to be caused by the conformational change involved in the cis–trans isomerization of the retinal chromophore. In contrast, the picosecond laser spectroscopy analysis of bovine rhodopsin at cryogenic temperatures revealed a large deuterium isotope effect and non-Arrhenius temperature dependence of the bathorhodopsin formation time, suggesting that the primary reaction corresponds to proton translocation.¹⁸ 

Retinal analogs with 11-cis- and all-trans-locked structures play key roles in determining the primary reactions in rhodopsins of animal and microbial origins, respectively. In the case of animal rhodopsins, 11-cis-locked retinals with five-, seven-, and eight-membered rings are incorporated into bovine opsin rendering the formed pigments unbleachable. Regardless of the absence of photobleaching, photophysical and photochemical property variations among the pigments were detected using picosecond time-resolved spectroscopy. No photoproduct was formed, and a long-lived excited state was observed for the five-membered-cycle rhodopsin (85 ps), which directly supported the isomerization model.¹⁹ Conversely, ground-state photoproducts were formed for seven- and eight-membered-cycle rhodopsins, returning to the original states within 100 ps and several nanoseconds, respectively.^19,20 These products resembled the photo-intermediates of bovine rhodopsin, photorhodopsin, and bathorhodopsin. Given the difference in rotational flexibilities along the C11=C12 double bond of the chromophore among the three locked analogs, the perceived correlation between the rhodopsin primary processes and C11=C12 bond flexibility supports the isomerization hypothesis. In fact, quantum chemical calculations on 11-cis-locked eight-membered-cycle rhodopsin reproduced the product state with an all-trans like structure.²¹ 

Unlike animal (visual) rhodopsins, which bleach after the 11-cis to all-trans photoisomerization, BR, a light-driven proton pump in purple membranes and the first discovered microbial rhodopsin, is nonbleachable upon illumination but photocyclable. Therefore, experiments on microbial rhodopsins can be conducted in the presence of light. This property of BR, together with its high thermal stability, has greatly assisted biophysical and biochemical investigations. Resonance Raman spectroscopy analysis showed that BR and the M intermediate, a key state in the proton pump, contain all-trans and 13-cis retinal residues, respectively.^22,23 The absorption of light by BR at 77 K induces the formation of a red-shifted K intermediate, which contained a 13-cis retinal chromophore as revealed by low-temperature Raman spectroscopy.²⁴ 

Therefore, at 77 K, BR possibly undergoes a shape-changing reaction involving the all-trans to 13-cis photoisomerization, which induces structural changes smaller [Fig. 2(b)] than the 11-cis to all-trans transformation [Fig. 2(a)]. However, the primary intermediate of BR is the J intermediate formed from the electronically excited state at <1 ps.^25,26 Hence, similar to animal rhodopsins, BR containing a C13=C14 trans–locked five-membered-ring retinal was synthesized and shown to exhibit a considerably prolonged excited-state lifetime (17–21 ps).²⁷ Although retinal chromophores with C13=C14 trans-locked rings of other sizes were not tested, this observation can be rationalized by the all-trans to 13-cis retinal isomerization in BR.

The above studies demonstrate that the primary photoreactions in both animal and microbial rhodopsins are retinal isomerizations. The fact that these isomerizations proceed even at low temperatures suggests that the corresponding structural changes are minor. To explain the unique retinal isomerization in proteins, several (e.g., sudden polarization,²⁸ bicycle pedal,²⁹ and hula twist³⁰) mechanistic models have been proposed. Vital insights have been provided by the comparison of isomerization pathways between protein and solution phases. High-performance liquid chromatography (HPLC) analysis showed that in the solution phase, the protonated Schiff base linkage of 11-cis retinal almost exclusively isomerizes into the all-trans form, suggesting that the reaction pathway in animal rhodopsin is due to the intrinsic properties of the chromophore.³¹ Conversely, in methanol, the protonated Schiff base linkage of all-trans retinal isomerizes into a mixture of cis products (82% 11-cis, 12% 9-cis, and 6% 13-cis).³¹ The formation of the 11-cis form as a photoproduct is an intrinsic property of retinochrome, an animal rhodopsin that serves as a retinal isomerase from all-trans into 11-cis forms.³² This fact highlights the role of protein environment of retinochrome in determining the features of the inherent pathway of the retinal chromophore photoisomerization, whereas in microbial rhodopsins, isomerization affords the 13-cis form. For both animal and microbial rhodopsins, the photoisomerization quantum yields are four to five folds higher than those in solution, indicating that the protein environment promotes retinal isomerization.⁷ 

To describe the detailed isomerization dynamics, one should capture the electronically excited states of rhodopsins, where the rotation around the double bond should take place. Given that picosecond resolution is insufficient for directly monitoring such processes, femtosecond laser pulse methods that emerged in the 1990s were applied to rhodopsins. The femtosecond transient absorption profile of bovine rhodopsin indicated that the generation of the all-trans form was completed within 200 fs.³³ The product formation kinetics were characterized by oscillatory features with a period of 550 fs, which were interpreted as coherent vibrational motions with a frequency of 60 cm⁻¹ in the isomerized product, and efficient photoisomerization was achieved by very rapid excited-state torsional motion.³⁴ Techniques such as femtosecond fluorescence spectroscopy³⁵ and femtosecond stimulated Raman spectroscopy³⁶ provide insights into the ultrafast photoisomerization in bovine rhodopsin. Based on these results, Fig. 3(a) presents the potential surfaces of the ground (S₀) and first excited (S₁) electronic states along the reaction coordinate (dihedral angle of the C11=C12 bond) for bovine rhodopsin. In the S₀ state, the energy barrier for the rotation around the C11=C12 bond is very high (>180 kJ/mol), whereas the measured thermal activation barrier is much lower (∼100 kJ/mol),^37,38 which suggests that thermal and light-driven processes take place via distinct pathways and implies that variations in local protein structures help lower the thermal activation barrier.³⁹ After photoexcitation to the Franck–Condon state, the excited wavepacket slides down the barrierless potential surface via chromophore twisting, traveling through the closest approach point of the energy surfaces, known as the conical intersection (CI).^40,41 Transient absorption spectroscopy with a sub-20 fs resolution was used to monitor the CI dynamics of bovine rhodopsin.⁴² In animal rhodopsins, the 11-cis to all-trans isomerization occurs with 100% selectivity, with the quantum yield for bovine rhodopsin determined as 0.67.⁴³ 

Regarding microbial rhodopsins, the femtosecond transient absorption spectroscopy analysis of BR showed that excited-state absorption is blue-shifted, and the primary J intermediate is red-shifted from the K intermediate.^25,26,33 The characteristic vibrational band of the 13-cis configuration at 1190 cm⁻¹ with a time constant of 500 fs observed by a femtosecond visible pump and IR probe spectroscopy indicated that the all-trans to 13-cis isomerization occurred on a femtosecond timescale.⁴⁴ The Fourier transform of the transient absorption data with <5 fs resolution exhibited the emergence of the 13-cis form in <1 ps, corroborating the above conclusion.⁴⁵ In addition, the J intermediate was shown by the anti-Stokes resonance Raman spectroscopy as a vibrationally hot state of the K intermediate, featuring a highly twisted and thermally excited chromophore.⁴⁶ As BR does not bleach, ultrafast spectroscopy was applied to various BR mutants to examine the roles of the surrounding residues. The replacement of the charged counterion residues decreased the photoisomerization rate and efficiency.⁴⁷ A detailed pH-dependent study of BR and proteorhodopsin revealed that the electrostatic interactions of the counterion complex in the ground state determine the primary reaction dynamics.⁴⁸ 

The potential surfaces of the S₀ and S₁ states along the dihedral angle of the C13=C14 bond in BR [Fig. 3(b)] resemble (but are not identical to) those of animal rhodopsins [Fig. 3(a)]. The selectivity and quantum yield of BR isomerization were determined as 100% and 0.64, respectively.^49,50 The three-state model of BR, as supported by several studies, resulted in a small potential barrier along the isomerization coordinate.^45,51–53 Conversely, the excited-state surface of the 13-cis forms of microbial rhodopsins exhibited ballistic barrierless wavepacket movement.^54,55

The occurrence of the shape-changing isomerization at cryogenic temperatures (e.g., 77 K) suggests that this transformation does not induce structural changes in the binding pocket of retinal. The femtosecond timescale of this reaction is fully consistent with this view, as the protein environment has no time for a large structural response. This suggests that the isomerized retinal chromophore (all-trans retinal in the binding pocket of 11-cis retinal in animal rhodopsins and 13-cis retinal in the binding pocket of all-trans retinal in microbial rhodopsins) is largely distorted. As revealed by low-temperature photocalorimetric studies, the structures of bathorhodopsin [Fig. 3(a)]⁵⁶ and K intermediate of BR [Fig. 3(b)]⁵⁷ store ∼60% (∼150 kJ/mol) and ∼30% (∼67 kJ/mol) of the light energy, respectively. Light energy dissipation by ∼70% is not problematic for BR, as the free energy required for proton pumping in this protein is ∼25 kJ/mol.⁵⁷ The results of monitoring increased hydrogen out-of-plane (HOOP) vibrational modes at 1000–800 cm⁻¹, energy is conserved in the highly twisted retinal chromophore. Interestingly, according to resonance Raman spectroscopy, the enhanced HOOP modes are localized at the center of the chromophore, e.g., at the C11=C12 bond in bovine rhodopsin,^58,59 and Schiff base region in BR.⁶⁰ 

Atomic-resolution structural information on rhodopsins is crucial for a more comprehensive understanding of the isomerization mechanism. Even though BR was the first membrane protein to have its structure deciphered by electron microscopy and found to comprise seven transmembrane helices,⁶¹ the structural determination of membrane proteins has not been easy in the last century. Eventually, techniques such as vibrational spectroscopy, including Raman and infrared spectroscopy, were used to analyze the structure of the retinal chromophore along with its interactions with the host protein.^62,63 The high-resolution structure of BR was first deciphered in 1998–1999 using lipidic cubic phase crystallization^64–66 and another method.⁶⁷ The crystal structure of bovine rhodopsin, the first G protein–coupled receptor subjected to structure determination, was deciphered in 2000.⁶⁸ 

The successful crystallization of rhodopsins enables the structural analysis of primary photo-intermediates, such as bathorhodopsin for animal rhodopsins and the K intermediate for microbial rhodopsins, by illuminating the crystals at low temperatures. Figure 4 compares the chromophore structures of rhodopsin⁶⁹ and bathorhodopsin at 105 K.⁷⁰ The corresponding electron densities overlap with each other despite the configurational differences (11-cis and all-trans, respectively). The change in the dihedral angle around the C11=C12 bond from 319° to 204° indicates isomerization (Fig. 5). Notably, a simple rotation around this bond results in the movement of either the β-ionone ring or the Schiff base. However, Fig. 4 shows minimal motion on either side. In fact, isomerization does not impact the strength of the hydrogen bonding of the Schiff base, which can be monitored using the difference between the C=NH and C=ND vibration frequencies⁷¹ and more directly using the N–D stretching frequency.⁷² This strongly suggests that light energy of ∼150 kJ/mol [Fig. 3(a)] is mainly stored in the distorted chromophore structure of bathorhodopsin. Recently, the isomerization dynamics of bovine rhodopsin have been directly examined at room temperature using time-resolved x-ray crystallography.⁷³ 

X ray crystallographic studies also revealed that isomerization does not significantly alter the protein structure of the primary K intermediate of BR^74–76 (Fig. 6). Figure 7 shows that in BR, during the all-trans to 13-cis isomerization, the Schiff base side is moved by the rotation around the C13=C14 bond. The lack of full rotation causes enhanced H–D exchangeable HOOP modes.⁶⁰ Water molecules act as acceptors of the hydrogen bonding of the protonated Schiff base in BR.^65–67 The analysis of the frequency difference between the C=NH and C=ND vibrations⁷⁷ and that of the N–D stretch⁷⁸ was used to monitor the weakened hydrogen bonding of the Schiff base upon the formation of the K intermediate. The emphasis was placed on weakening the hydrogen bonds of water molecules by retinal isomerization in BR,^79,80 which may contribute to light energy storage. The necessity of altering hydrogen bonding for energy storage highlights that the strong hydrogen bonding of water near the Schiff base is positively correlated with proton pumping activity.^81,82 Ultrafast isomerization dynamics in BR at room temperature were observed by time-resolved x-ray crystallography.^83,84

Computational science has enabled considerable progress by helping determine the atomic coordinates of animal and microbial rhodopsins, while quantum mechanics and molecular mechanics methods have been used to describe the primary reaction dynamics.^85–96 To explain the space-saving barrierless isomerization in rhodopsins, space-saving reaction coordinates were computationally searched in the excited state. Inspired by the initial bicycle pedal model,²⁹ researchers proposed aborted bicycle pedal coordinates for bovine rhodopsin,⁴¹ with the HOOP wag motions of the retinal chromophore playing a crucial role in efficient isomerization. The structural changes in bovine rhodopsin calculated in 2011 (Ref. 90) well match those determined by time-resolved x-ray crystallography in 2023.⁷³ 

Chromophore distortion can be predominantly attributed to primary intermediates formed by retinal isomerization having energy levels considerably higher than the initial resting state.^56,57 In addition to chromophore distortion, the weakened hydrogen bonds in the K intermediate of BR can also store energy, featuring an energy storage capacity (46 kJ/mol)^97,98 accounting for more than half of the total (67 kJ/mol). Upon light absorption on a timescale of 10⁻¹⁵ s, photoisomerization occurs in 10⁻¹⁴–10⁻¹² s, with light energy stored as steric limitations in animal rhodopsins or hydrogen-bonding alterations and steric hindrances in microbial rhodopsins. The above-mentioned studies focused on bovine rhodopsin and BR but may also be performed for other animal and microbial rhodopsins.

All rhodopsins undergo photoisomerization at low temperatures, with 11-cis to all-trans and all-trans to 13-cis isomerizations occurring in animal and microbial rhodopsins, respectively. Extensive studies using spectroscopic techniques, structural biology, and theoretical calculations have elucidated the mechanism of these unique isomerizations. Based on these properties, protein-bound water molecules were systematically analyzed by Fourier transform infrared spectroscopy at 77 K.⁸² Recently discovered microbial rhodopsins, neorhodopsin (NeoR), isolated in 2020 from Rhizoclosmatium globosum, and bestrhodopsin, isolated in 2022 from marine unicellular algae, exhibit exceptional photochemical properties. Specifically, the photoreactions of these species do not proceed at low temperatures and do not afford the 13-cis form.

The protonated Schiff base of all-trans retinal features an absorption maximum (λ_max) of 610 nm in the gas phase,⁹⁹ i.e., the λ_max of rhodopsin should only be visible at <610 nm. However, NeoR, a near-infrared light–absorbing rhodopsin with guanylyl cyclase activity,¹⁰⁰ has λ_max ≈ 690 nm [Fig. 8(a)]. Bestrhodopsin contains one or two rhodopsin domains fused to a bestrophin channel that serves as a light-gated ion channel.¹⁰¹  Figure 8(a) shows that the two rhodopsin domains in bestrhodopsin feature λ_max ≈ 660 nm. The unusually red-shifted absorption of bestrhodopsin and NeoR is due to a unique counterion triad composed of carboxylic acids, two in the C-helix and one in the G-helix.^100,101 Although the color-tuning mechanism is intriguing, the corresponding photochemistry is unusual.

Bestrhodopsin does not undergo an all-trans to 11-cis photoisomerization at 77–150 K (Ref. 101) and is therefore the first rhodopsin without any photoreaction in this temperature range. Retinochrome, an animal rhodopsin acting as a retinal isomerase,³² undergoes a similar photoisomerization even at 77 K.¹⁰² Moreover, NeoR isolated from Obelidium mucronatum undergoes an all-trans to 7-cis photoisomerization.¹⁰³ The steric conflict between the 9-methyl group and the methyl groups in the β-ionone ring destabilizes 7-cis retinal; nevertheless, the presence of photoproducts containing the 7-cis form was confirmed by HPLC and nuclear magnetic resonance spectroscopy. Although the photoproduct of NeoR in the 7-cis form was stable at room temperature, the all-trans to 7-cis photoisomerization was prevented at <270 K.

The absence of photoreactions at low temperatures implies the presence of high potential barriers in the excited state, unlike those of bovine rhodopsin and BR (Fig. 3). As described previously, the protonated Schiff base of all-trans retinal in the solution phase is mainly isomerized into the 11-cis form,³¹ which may indicate that the specific chromophore–protein interaction in these microbial rhodopsins is destroyed. However, the cryo-electron microscopic structure of bestrhodopsin resembles those of other microbial rhodopsins, although the chromophore of the former is considerably distorted.¹⁰¹ Thus, one should determine why the “normal” isomerization into the 13-cis form is prohibited in these proteins. As described above, the ionone ring side is fixed in microbial rhodopsins, and the Schiff base side rotates. So, what is the structure of the 7-cis product formed in the case of NeoR? Similar to other microbial rhodopsins, NeoR features an s-trans C6–C7 bond, which means that the 7-cis form would suffer from steric hindrance between the 9-methyl group and the two methyl groups of the β-ionone ring. The molecular mechanism of the exceptional photoisomerizations of bestrhodopsin and NeoR should be clarified in the future, and the unique features of the excited states in NeoR have been recently reported.^104–106 

Isomerization is a shape-changing reaction that is not expected to occur at low temperatures. Given that the primary photo-intermediates of rhodopsins form even at 77 K, retinal isomerization has been questioned as the primary reaction in rhodopsins. However, long-standing experimental efforts, mainly involving spectroscopic methods, have led to the conclusion that this reaction is indeed an isomerization. Rhodopsins probably engage in a shape-changing reaction without altering the shape of their retinal binding pocket, with the aborted bicycle pedal mechanism contributing to the space-saving reaction. Animal and microbial rhodopsins follow different mechanisms despite the few common reactions involved. In animal rhodopsins, only the central part is changed by isomerization at C11=C12, while the strength of hydrogen bonding involving the protonated Schiff base remains constant. Consequently, light energy is stored exclusively in the form of retinal distortion. In microbial rhodopsins, isomerization at the C13=C14 bond changes the hydrogen bonding in the Schiff base region and weakens the hydrogen bonds of the protein-bound water molecules. Consequently, light energy is stored in the forms of retinal distortion and weakened hydrogen bond network. Recently, unusual isomerization pathways (all-trans to 7-cis or 11-cis) and temperature effects (no reactions at <273  or <170 K) have been reported for near-infrared light–absorbing microbial rhodopsins, such as bestrhodopsin and NeoR.

The authors have no conflicts to disclose.

Hideki Kandori: Conceptualization (lead); Funding acquisition (lead); Writing – original draft (lead). Masahiro Sugiura: Writing – review & editing (equal). Kota Katayama: Writing – review & editing (equal).

The data that support the findings of this study are available from the corresponding author upon reasonable request.