The retinal chromophore environment in an inward light-driven proton pump studied by solid-state NMR and hydrogen-bond network analysis.

Inward proton pumping is a relatively new function for microbial rhodopsins, retinal-binding light-driven membrane proteins. So far, it has been demonstrated for two unrelated subgroups of microbial rhodopsins, xenorhodopsins and schizorhodopsins. A number of recent studies suggest unique retinal-protein interactions as being responsible for the reversed direction of proton transport in the latter group. Here, we use solid-state NMR to analyze the retinal chromophore environment and configuration in an inward proton-pumping Antarctic schizorhodopsin. Using fully 13C-labeled retinal, we have assigned chemical shifts for every carbon atom and, assisted by structure modelling and molecular dynamics simulations, made a comparison with well-studied outward proton pumps, identifying locations of the unique protein-chromophore interactions for this functional subclass of microbial rhodopsins. Both the NMR results and molecular dynamics simulations point to the distinctive polar environment in the proximal part of the retinal, which may result in a hydration pattern dramatically different from that of the outward proton pumps, causing the reversed proton transport.

Structural insights into the mechanism and dynamics of proteorhodopsin biogenesis and retinal scavenging.

Microbial ion-pumping rhodopsins (MRs) are extensively studied retinal-binding membrane proteins. However, their biogenesis, including oligomerisation and retinal incorporation, remains poorly understood. The bacterial green-light absorbing proton pump proteorhodopsin (GPR) has emerged as a model protein for MRs and is used here to address these open questions using cryo-electron microscopy (cryo-EM) and molecular dynamics (MD) simulations. Specifically, conflicting studies regarding GPR stoichiometry reported pentamer and hexamer mixtures without providing possible assembly mechanisms. We report the pentameric and hexameric cryo-EM structures of a GPR mutant, uncovering the role of the unprocessed N-terminal signal peptide in the assembly of hexameric GPR. Furthermore, certain proteorhodopsin-expressing bacteria lack retinal biosynthesis pathways, suggesting that they scavenge the cofactor from their environment. We shed light on this hypothesis by solving the cryo-EM structure of retinal-free proteoopsin, which together with mass spectrometry and MD simulations suggests that decanoate serves as a temporary placeholder for retinal in the chromophore binding pocket. Further MD simulations elucidate possible pathways for the exchange of decanoate and retinal, offering a mechanism for retinal scavenging. Collectively, our findings provide insights into the biogenesis of MRs, including their oligomeric assembly, variations in protomer stoichiometry and retinal incorporation through a potential cofactor scavenging mechanism.

Low-temperature FTIR spectroscopy of the L/Q switch of proteorhodopsin.

Rhodopsins are photoreceptive membrane proteins containing a retinal chromophore, and the color tuning mechanism in rhodopsins is one of the important topics. Color switch is a color-determining residue at the same position, where replacement of red- and blue-shifting amino acids in two wild-type rhodopsins causes spectral blue- and red-shifts, respectively. The first and most famous color switch in microbial rhodopsins is the L/Q switch in proteorhodopsins (PRs). Green- or blue-absorbing PR (GPR or BPR) contains Leu and Gln at position 105 of the C-helix (TM3), respectively, and their replacement converted absorbing colors. The L/Q switch enables bacteria to absorb green or blue light in shallow or deep ocean waters, respectively. Although Gln and Leu are hydrophilic and hydrophobic residues, respectively, a comprehensive mutation study of position 105 in GPR revealed that the λmax correlated with the volume of residues, not the hydropathy index. To gain structural insights into the mechanism, we applied low-temperature FTIR spectroscopy of L105Q GPR, and the obtained spectra were compared with those of GPR and BPR. The difference FTIR spectra of L105Q GPR were similar to those of BPR, not GPR, implying that the L/Q switch converts the GPR structure into a BPR structure in terms of the local environments of the retinal chromophore. It includes retinal skeletal vibration, hydrogen-bonding strength of the protonated Schiff base, amide-A vibration (peptide backbone), and protein-bound water molecules. Consequently color is switched accompanying such structural alterations, and known as the L/Q switch.

Contribution of Proteorhodopsin to Light-Dependent Biological Responses in Hymenobacter nivis P3T Isolated from Red Snow in Antarctica.

Proteorhodopsin (PR) is a major family of microbial rhodopsins that function as light-driven outward proton pumps. PR is now widely recognized for its ecological importance as a molecule responsible for solar energy flow in various ecosystems on the earth. However, few concrete examples of the actual use of light by natural microorganisms via PR have been demonstrated experimentally. This study reveals one example of that in a cryophilic bacterium Hymenobacter nivis P3T isolated from red snow in Antarctica. The results demonstrate light-dependent biochemical and biological responses in H. nivis cells, such as the proton pump activity of H. nivis PR (HnPR), which leads to the production of proton motive force, cellular ATP production, and cell growth. In addition, the results of this study demonstrate the photochemical properties of a PR, namely, HnPR, in the membrane of a natural host bacterium. The photocycle of HnPR was much faster than other PRs even at 5 °C, indicating that the proton pump function of HnPR has adapted to the low-temperature environment of Antarctica. Although it is well-known that PR helps natural host microorganisms to use light energy, this study provides another concrete example for understanding the biological role of PR by demonstrating the link between the molecular functions of PR and the light-dependent biochemical and biological responses of a PR-bearing host.

Unusual Vibrational Coupling of the Schiff Base in the Retinal Chromophore of Sodium Ion-Pumping Rhodopsins.

A Schiff base in the retinal chromophore of microbial rhodopsin is crucial to its ion transport mechanism. Here, we discovered an unprecedented isotope effect on the C═N stretching frequency of the Schiff base in sodium ion-pumping rhodopsins, showing an unusual interaction of the Schiff base. No amino acid residue attributable to the unprecedented isotope effect was identified, suggesting that the H-O-H bending vibration of a water molecule near the Schiff base was coupled with the C═N stretching vibration. A twist in the polyene chain in the chromophore for the sodium ion-pumping rhodopsins enabled this unusual interaction of the Schiff base. The present discovery provides new insights into the interaction network of the retinal chromophore in microbial rhodopsins.

Unveiling the critical role of K+ for xanthorhodopsin expression in E. coli.

Xanthorhodopsin (XR), a retinal-binding 7-transmembrane protein isolated from the eubacterium Salinibacter ruber, utilizes two chromophores (retinal and salinixanthin (SAL)) as an outward proton pump and energy-donating carotenoid. However, research on XR has been impeded owing to limitations in achieving heterogeneous expression of stable forms and high production levels of both wild-type and mutants. We successfully expressed wild-type and mutant XRs in Escherichia coli in the presence of K+. Achieving XR expression requires significant K+ and a low inducer concentration. In particular, we highlight the significance of Ser-159 in helix E located near Gly-156 (a carotenoid-binding position) as a critical site for XR expression. Our findings indicate that replacing Ser-159 with a smaller amino acid, alanine, can enhance XR expression in a manner comparable to K+, implying that Ser-159 poses a steric hindrance for pigment formation in XR. In the presence of K+, the proton pumping and photocycle of the wild-type and mutants were characterized and compared; the wild-type result suggests similar properties to the first reported XR isolation from the S. ruber membrane fraction. We propose that the K+ gradient across the cell membrane of S. ruber serves to uphold the membrane potential of the organism and plays a role in the expression of proteins, such as XR, as demonstrated in our study. Our findings deepen the understanding of adaptive protein expression, particularly in halophilic organisms. We highlight salt selection as a promising strategy for improving protein yield and functionality.

Hydrogen-Bonding and Hydrophobic Interaction Networks as Structural Determinants of Microbial Rhodopsin Function.

Microbial pump rhodopsins are highly versatile light-driven membrane proteins that couple protein conformational dynamics with ion translocation across the cell membranes. Understanding how microbial pump rhodopsins use specific amino acid residues at key functional sites to control ion selectivity and ion pumping direction is of general interest for membrane transporters, and could guide site-directed mutagenesis for optogenetics applications. To enable direct comparisons between proteins with different sequences we implement, for the first time, a unique numbering scheme for the microbial pump rhodopsin residues, NS-mrho. We use NS-mrho to show that distinct microbial pump rhodopsins typically have hydrogen-bond networks that are less conserved than anticipated from the amino acid residue conservation, whereas their hydrophobic interaction networks are largely conserved. To illustrate the role of the hydrogen-bond networks as structural elements that determine the functionality of microbial pump rhodopsins, we performed experiments, atomic-level simulations, and hydrogen bond network analyses on GR, the outward proton pump from Gloeobacter violaceus, and KR2, the outward sodium pump from Krokinobacter eikastus. The experiments indicate that multiple mutations that recover KR2 amino acid residues in GR not only fail to convert it into a sodium pump, but completely inactivate GR by abolishing photoisomerization of the retinal chromophore. This observation could be attributed to the drastically altered hydrogen-bond interaction network identified with simulations and network analyses. Taken together, our findings suggest that functional specificity could be encoded in the collective hydrogen-bond network of microbial pump rhodopsins.

Calcium Binding Mechanism in TAT Rhodopsin.

TAT rhodopsin binds Ca2+ near the Schiff base region, which accompanies deprotonation of the Schiff base. This paper reports the Ca2+-free and Ca2+-bound structures of TAT rhodopsin by molecular dynamics (MD) simulation launched from AlphaFold structures. In the Ca2+-bound TAT rhodopsin, Ca2+ is directly coordinated by eight oxygen atoms, four oxygens of the side chains of E54 and D227, and four oxygens of water molecules. E54 is not involved in the hydrogen-bonding network of the Ca2+-free TAT rhodopsin, while flipping motion of E54 allows Ca2+ binding to TAT rhodopsin with deformation of helices observed by FTIR spectroscopy. The hydrogen-bonding network plays a crucial role in maintaining the Ca2+ binding, as mutations of E54, Y55, R79, Y200, E220, and D227 abolished the binding. Only T82V exhibited the Ca2+ binding like the wild type among the mutants in this study. The molecular mechanism of Ca2+ binding is discussed based on the present computational and experimental analysis.

Bidirectional Optical Control of Proton Motive Force in Escherichia coli Using Microbial Rhodopsins.

Proton (H+) motive force (PMF) serves as the energy source for the flagellar motor rotation, crucial for microbial motility. Here, to control PMF using light, we introduced light-driven inward and outward proton pump rhodopsins, RmXeR and AR3, into Escherichia coli. The motility of E. coli cells expressing RmXeR and AR3 significantly decreased and increased upon illumination, respectively. Tethered cell experiments revealed that, upon illumination, the torque of the flagellar motor decreased to nearly zero (28 pN nm) with RmXeR, while it increased to 1170 pN nm with AR3. These alterations in PMF correspond to +146 mV (RmXeR) and -140 mV (AR3), respectively. Thus, bidirectional optical control of PMF in E. coli was successfully achieved by using proton pump rhodopsins. This system holds a potential for enhancing our understanding of the roles of PMF in various biological functions.

SNap Bond, a Crucial Hydrogen Bond Between Ser in Helix 3 and Asn in Helix 4, Regulates the Structural Dynamics of Heliorhodopsin.

Heliorhodopsin (HeR) is a new rhodopsin family discovered in 2018 through functional metagenomic analysis. Similar to microbial rhodopsins, HeR has an all-trans retinal chromophore, and its photoisomerization to the 13-cis form triggers a relatively slow photocycle with sequential intermediate states (K, M, and O intermediates). The O intermediate has a relatively long lifetime and is a putative active state for transferring signals or regulating enzymatic reactions. Although the first discovered HeR, 48C12, was found in bacteria and the second HeR (TaHeR) was found in archaea, their key amino acid residues and molecular architectures have been recognized to be well conserved. Nevertheless, the rise and decay kinetics of the O intermediate are faster in 48C12 than in TaHeR. Here, using a new infrared spectroscopic technique with quantum cascade lasers, we clarified that the hydrogen bond between transmembrane helices (TM) 3 and 4 is essential for the altered O kinetics (Ser112 and Asn138 in 48C12). Interconverting mutants of 48C12 and TaHeR clearly revealed that the hydrogen bond is important for regulating the dynamics of the O intermediate. Overall, our study sheds light on the importance of the hydrogen bond between TM3 and TM4 in heliorhodopsins, similar to the DC gate in channelrhodopsins.

Excited-State Dynamics of a CRABPII-Based Microbial Rhodopsin Mimic.

Microbial rhodopsin, a pivotal photoreceptor protein, has garnered widespread application in diverse fields such as optogenetics, biotechnology, biodevices, etc. However, current microbial rhodopsins are all transmembrane proteins, which both complicates the investigation on the photoreaction mechanism and limits their further applications. Therefore, a specific mimic for microbial rhodopsin can not only provide a better model for understanding the mechanism but also can extend the applications. The human protein CRABPII turns out to be a good template for design mimics on rhodopsin due to the convenience in synthesis and the stability after mutations. Recently, Geiger et al. designed a new CRABPII-based mimic M1-L121E on microbial rhodopsin with the 13-cis, syn (13C) isomerization after irradiation. However, it still remains a question as to how similar it is compared with the natural microbial rhodopsin, in particular, in the aspect of the photoreaction dynamics. In this article, we investigate the excited-state dynamics of this mimic by measuring its transient absorption spectra. Our results reveal that there are two components in the solution of mimic M1-L121E at pH 8, known as protonated Schiff base (PSB) and unprotonated Schiff base (USB) states. In both states, the photoreaction process from 13-cis, syn(13C) to all-trans,anti (AT) is faster than that from the inverse direction. In addition, the photoreaction process in the PSB state is faster than that in the USB state. We compared the isomerization time of the PSB state to that of microbial rhodopsin. Our findings indicate that M1-L121E exhibits behaviors similar to those of microbial rhodopsins in the general pattern of PSB isomerization, where the isomerization from 13C to AT is much faster than its inverse direction. However, our results also reveal significant differences in the excited-state dynamics of the mimic relative to the native microbial rhodopsin, including the slower PSB isomerization rates as well as the unusual USB photoreaction dynamics at pH = 8. By elucidating the distinctive characteristics of mimics M1-L121E, this study enhances our understanding of microbial rhodopsin mimics and their potential applications.

Photoisomerization pathway of the microbial rhodopsin chromophore in solution.

Photoisomerization is a key photochemical reaction in microbial and animal rhodopsins. It is well established that such photoisomerization is highly selective; all-trans to 13-cis, and 11-cis to all-trans forms in microbial and animal rhodopsins, respectively. Nevertheless, unusual photoisomerization pathways have been discovered recently in microbial rhodopsins. In an enzymerhodopsin NeoR, the all-trans chromophore is isomerized into the 7-cis form exclusively, which is stable at room temperature. Although, the 7-cis form is produced by illumination of retinal, formation of the 7-cis form was never reported for a protonated Schiff base of all-trans retinal in solution. Present HPLC analysis of retinal oximes prepared by hydroxylamine reaction revealed that all-trans and 7-cis forms cannot be separated from the syn peaks under the standard HPLC conditions, while it is possible by the analysis of the anti-peaks. Consequently, we found formation of the 7-cis form by the photoreaction of all-trans chromophore in solution, regardless of the protonation state of the Schiff base. Upon light absorption of all-trans protonated retinal Schiff base in solution, excited-state relaxation accompanies double-bond isomerization, producing 7-cis, 9-cis, 11-cis, or 13-cis form. In contrast, specific chromophore-protein interaction enforces selective isomerization into the 13-cis form in many microbial rhodopsins, but into 7-cis in NeoR.

RhoMax: Computational Prediction of Rhodopsin Absorption Maxima Using Geometric Deep Learning.

Microbial rhodopsins (MRs) are a diverse and abundant family of photoactive membrane proteins that serve as model systems for biophysical techniques. Optogenetics utilizes genetic engineering to insert specialized proteins into specific neurons or brain regions, allowing for manipulation of their activity through light and enabling the mapping and control of specific brain areas in living organisms. The obstacle of optogenetics lies in the fact that light has a limited ability to penetrate biological tissues, particularly blue light in the visible spectrum. Despite this challenge, most optogenetic systems rely on blue light due to the scarcity of red-shifted opsins. Finding additional red-shifted rhodopsins would represent a major breakthrough in overcoming the challenge of limited light penetration in optogenetics. However, determining the wavelength absorption maxima for rhodopsins based on their protein sequence is a significant hurdle. Current experimental methods are time-consuming, while computational methods lack accuracy. The paper introduces a new computational approach called RhoMax that utilizes structure-based geometric deep learning to predict the absorption wavelength of rhodopsins solely based on their sequences. The method takes advantage of AlphaFold2 for accurate modeling of rhodopsin structures. Once trained on a balanced train set, RhoMax rapidly and precisely predicted the maximum absorption wavelength of more than half of the sequences in our test set with an accuracy of 0.03 eV. By leveraging computational methods for absorption maxima determination, we can drastically reduce the time needed for designing new red-shifted microbial rhodopsins, thereby facilitating advances in the field of optogenetics.

Energetics of the H-Bond Network in Exiguobacterium sibiricum Rhodopsin.

Exiguobacterium sibiricum rhodopsin (ESR) functions as a light-driven proton pump utilizing Lys96 for proton uptake and maintaining its activity over a wide pH range. Using a combination of methodologies including the linear Poisson-Boltzmann equation and a quantum mechanical/molecular mechanical approach with a polarizable continuum model, we explore the microscopic mechanisms underlying its pumping activity. Lys96, the primary proton uptake site, remains deprotonated owing to the loss of solvation in the ESR protein environment. Asp85, serving as a proton acceptor group for Lys96, does not form a low-barrier H-bond with His57. Instead, deprotonated Asp85 forms a salt-bridge with protonated His57, and the proton is predominantly located at the His57 moiety. Glu214, the only acidic residue at the end of the H-bond network exhibits a pKa value of ∼6, slightly elevated due to solvation loss. It seems likely that the H-bond network [Asp85···His57···H2O···Glu214] serves as a proton-conducting pathway toward the protein bulk surface.

Calcium-Sensitive Microbial Rhodopsin VirChR1: A Femtosecond to Second Photocycle Study.

Viral rhodopsins are light-gated cation channels representing a novel class of microbial rhodopsins. For viral rhodopsin 1 subfamily members VirChR1 and OLPVR1, channel activity is abolished above a certain calcium concentration. Here we present a calcium-dependent spectroscopic analysis of VirChR1 on the femtosecond to second time scale. Unlike channelrhodopsin-2, VirChR1 possesses two intermediate states P1 and P2 on the ultrafast time scale, similar to J and K in ion-pumping rhodopsins. Subsequently, we observe multifaceted photocycle kinetics with up to seven intermediate states. Calcium predominantly affects the last photocycle steps, including the appearance of additional intermediates P6Ca and P7 representing the blocked channel. Furthermore, the photocycle of the counterion variant D80N is drastically altered, yielding intermediates with different spectra and kinetics compared to those of the wt. These findings demonstrate the central role of the counterion within the defined reaction sequence of microbial rhodopsins that ultimately defines the protein function.

Heliorhodopsin-mediated light-modulation of ABC transporter.

Heliorhodopsins (HeRs) have been hypothesized to have widespread functions. Recently, the functions for few HeRs have been revealed; however, the hypothetical functions remain largely unknown. Herein, we investigate light-modulation of heterodimeric multidrug resistance ATP-binding cassette transporters (OmrDE) mediated by Omithinimicrobium cerasi HeR. In this study, we classifiy genes flanking the HeR-encoding genes and identify highly conservative residues for protein-protein interactions. Our results reveal that the interaction between OcHeR and OmrDE shows positive cooperatively sequential binding through thermodynamic parameters. Moreover, light-induced OcHeR upregulates OmrDE drug transportation. Hence, the binding may be crucial to drug resistance in O. cerasi as it survives in a drug-containing habitat. Overall, we unveil a function of HeR as regulatory rhodopsin for multidrug resistance. Our findings suggest potential applications in optogenetic technology.

Picosecond quantum-classical dynamics reveals that the coexistence of light-induced microbial and animal chromophore rotary motion modulates the isomerization quantum yield of heliorhodopsin.

Rhodopsins are light-responsive proteins forming two vast and evolutionary distinct superfamilies whose functions are invariably triggered by the photoisomerization of a single retinal chromophore. In 2018 a third widespread superfamily of rhodopsins called heliorhodopsins was discovered using functional metagenomics. Heliorhodopsins, with their markedly different structural features with respect to the animal and microbial superfamilies, offer an opportunity to study how evolution has manipulated the chromophore photoisomerization to achieve adaptation. One question is related to the mechanism of such a reaction and how it differs from that of animal and microbial rhodopsins. To address this question, we use hundreds of quantum-classical trajectories to simulate the spectroscopically documented picosecond light-induced dynamics of a heliorhodopsin from the archaea thermoplasmatales archaeon (TaHeR). We show that, consistently with the observations, the trajectories reveal two excited state decay channels. However, inconsistently with previous hypotheses, only one channel is associated with the -C13C14- rotation of microbial rhodopsins while the second channel is characterized by the -C11C12- rotation typical of animal rhodopsins. The fact that such -C11C12- rotation is aborted upon decay and ground state relaxation, explains why illumination of TaHeR only produces the 13-cis isomer with a low quantum efficiency. We argue that the documented lack of regioselectivity in double-bond excited state twisting motion is the result of an "adaptation" that could be completely lost via specific residue substitutions modulating the steric hindrance experienced along the isomerization motion.

Multiple Roles of a Conserved Glutamate Residue for Unique Biophysical Properties in a New Group of Microbial Rhodopsins Homologous to TAT Rhodopsin.

TAT rhodopsin, a microbial rhodopsin found in the marine SAR11 bacterium HIMB114, uniquely possesses a Thr-Ala-Thr (TAT) motif in the third transmembrane helix. Because of a low pKa value of the retinal Schiff base (RSB), TAT rhodopsin exhibits both a visible light-absorbing state with the protonated RSB and a UV-absorbing state with the deprotonated RSB at a neutral pH. The UV-absorbing state, in contrast to the visible light-absorbing one, converts to a long-lived photointermediate upon light absorption, implying that TAT rhodopsin functions as a pH-dependent light sensor. Despite detailed biophysical characterization and mechanistic studies on the TAT rhodopsin, it has been unknown whether other proteins with similarly unusual features exist. Here, we identified several new rhodopsin genes homologous to the TAT rhodopsin of HIMB114 (TATHIMB) from metagenomic data. Based on the absorption spectra of expressed proteins from these genes with visible and UV peaks similar to that of TATHIMB, they were classified as Twin-peaked Rhodopsin (TwR) family. TwR genes form a gene cluster with a set of 13 ORFs conserved in subclade IIIa of SAR11 bacteria. A glutamic acid in the second transmembrane helix, Glu54, is conserved in all of the TwRs. We investigated E54Q mutants of two TwRs and revealed that Glu54 plays critical roles in regulating the RSB pKa, oligomer formation, and the efficient photoreaction of the UV-absorbing state. The discovery of novel TwRs enables us to study the universality and individuality of the characteristics revealed so far in the original TATHIMB and contributes to further studies on mechanisms of unique properties of TwRs.

Internal Proton Transfer in the Activation of Heliorhodopsin.

Heliorhodopsin (HeR), a recently discovered new rhodopsin family, contains a single counterion of the protonated Schiff base, E108 in HeR from Thermoplasmatales archaeon SG8-52-1 (TaHeR). Upon light absorption, the M and O intermediates form in HeRs, as well as type-1 microbial rhodopsins, indicating that the proton transfer from the Schiff base leads to the activation of HeRs. The present flash photolysis study of TaHeR in the presence of a pH-sensitive dye showed that TaHeR contains a proton-accepting group (PAG) inside protein. Comprehensive mutation study of TaHeR found the E108D mutant abolishing the M formation, which is not only at pH 8, but also at pH 9 and 10. The lack of M observation does not originate from the short lifetime of the M intermediate in E108D, as FTIR spectroscopy revealed that a red-shifted K-like intermediate is long lived in E108D. It is likely that the K-like intermediate returns to the unphotolyzed state without internal proton transfer in E108D. E108 and D108 are the Schiff base counterions of the wild-type and E108D mutant TaHeR, respectively, whereas small difference in length of side chains determine internal proton transfer reaction from the Schiff base. Based on the present finding, we propose that the internal water cluster (four water molecules) constitutes PAG in the M intermediate of TaHeR. In the wild type TaHeR, a protonated water cluster is stabilized by forming a salt bridge with E108. In contrast, slightly shortened counterion (D108) cannot stabilize the protonated water cluster in E108D, and thus impairs internal proton transfer from the Schiff base.

Experimental Assessment of the Electronic and Geometrical Structure of a Near-Infrared Absorbing and Highly Fluorescent Microbial Rhodopsin.

The recently discovered Neorhodopsin (NeoR) exhibits absorption and emission maxima in the near-infrared spectral region, which together with the high fluorescence quantum yield makes it an attractive retinal protein for optogenetic applications. The unique optical properties can be rationalized by a theoretical model that predicts a high charge transfer character in the electronic ground state (S0) which is otherwise typical of the excited state S1 in canonical retinal proteins. The present study sets out to assess the electronic structure of the NeoR chromophore by resonance Raman (RR) spectroscopy since frequencies and relative intensities of RR bands are controlled by the ground and excited state's properties. The RR spectra of NeoR differ dramatically from those of canonical rhodopsins but can be reliably reproduced by the calculations carried out within two different structural models. The remarkable agreement between the experimental and calculated spectra confirms the consistency and robustness of the theoretical approach.