Chromophore-Protein Interactions Affecting the Polyene Twist and π-π* Energy Gap of the Retinal Chromophore in Schizorhodopsins.

The properties of a prosthetic group are broadened by interactions with its neighboring residues in proteins. The retinal chromophore in rhodopsins absorbs light, undergoes structural changes, and drives functionally important structural changes in proteins during the photocycle. It is therefore crucial to understand how chromophore-protein interactions regulate the molecular structure and electronic state of chromophores in rhodopsins. Schizorhodopsin is a newly discovered subfamily of rhodopsins found in the genomes of Asgard archaea, which are extant prokaryotes closest to the last common ancestor of eukaryotes and of other microbial species. Here, we report the effects of a hydrogen bond between a retinal Schiff base and its counterion on the twist of the polyene chain and the color of the retinal chromophore. Correlations between spectral features revealed the unexpected fact that the twist of the polyene chain is reduced as the hydrogen bond becomes stronger, suggesting that the twist is caused by tight atomic contacts between the chromophore and nearby residues. In addition, the strength of the hydrogen bond is the primary factor affecting the color-tuning of the retinal chromophore in schizorhodopsins. The findings of this study are valuable for manipulating the molecular structure and electronic state of the chromophore by controlling chromophore-protein interactions.

Cis-Trans Reisomerization Preceding Reprotonation of the Retinal Chromophore Is Common to the Schizorhodopsin Family: A Simple and Rational Mechanism for Inward Proton Pumping.

The creation of unidirectional ion transporters across membranes represents one of the greatest challenges in chemistry. Proton-pumping rhodopsins are composed of seven transmembrane helices with a retinal chromophore bound to a lysine side chain via a Schiff base linkage and provide valuable insights for designing such transporters. What makes these transporters particularly intriguing is the discovery of both outward and inward proton-pumping rhodopsins. Surprisingly, despite sharing identical overall structures and membrane topologies, these proteins facilitate proton transport in opposite directions, implying an underlying rational mechanism that can transport protons in different directions within similar protein structures. In this study, we unraveled this mechanism by examining the chromophore structures of deprotonated intermediates in schizorhodopsins, a recently discovered subfamily of inward proton-pumping rhodopsins, using time-resolved resonance Raman spectroscopy. The photocycle of schizorhodopsins revealed the cis-trans thermal isomerization that precedes reprotonation at the Schiff base of the retinal chromophore. Notably, this order has not been observed in other proton-pumping rhodopsins, but here, it was observed in all seven schizorhodopsins studied across the archaeal domain, strongly suggesting that cis-trans thermal isomerization preceding reprotonation is a universal feature of the schizorhodopsin family. Based on these findings, we propose a structural basis for the remarkable order of events crucial for facilitating inward proton transport. The mechanism underlying inward proton transport by schizorhodopsins is straightforward and rational. The insights obtained from this study hold great promise for the design of transmembrane unidirectional ion transporters.

Unique Vibrational Characteristics and Structures of the Photoexcited Retinal Chromophore in Ion-Pumping Rhodopsins.

Photoisomerization of an all-trans-retinal chromophore triggers ion transport in microbial ion-pumping rhodopsins. Understanding chromophore structures in the electronically excited (S1) state provides insights into the structural evolution on the potential energy surface of the photoexcited state. In this study, we examined the structure of the S1-state chromophore in Natronomonas pharaonis halorhodopsin (NpHR), a chloride ion-pumping rhodopsin, using time-resolved resonance Raman spectroscopy. The spectral patterns of the S1-state chromophore were completely different from those of the ground-state chromophore, resulting from unique vibrational characteristics and the structure of the S1 state. Mode assignments were based on a combination of deuteration shifts of the Raman bands and hybrid quantum mechanics-molecular mechanics calculations. The present observations suggest a weakened bond alternation in the π conjugation system. A strong hydrogen-out-of-plane bending band was observed in the Raman spectra of the S1-state chromophore in NpHR, indicating a twisted polyene structure. Similar frequency shifts for the C═N/C═C and C-C stretching modes of the S1-state chromophore in NpHR were observed in the Raman spectra of sodium ion-pumping and proton-pumping rhodopsins, suggesting that these unique features are common to the S1 states of ion-pumping rhodopsins.

Heme Pocket Structure and Its Functional Implications in an Ancestral Globin Protein.

Proteins have undergone evolutionary processes to achieve optimal stability, increased functionality, and novel functions. Comparative analysis of existent and ancestral proteins provides insights into the factors that influence protein stability and function. Ancestral sequence reconstruction allows us to deduce the amino acid sequences of ancestral proteins. Here, we present the structural and functional characteristics of an ancestral protein, AncMH, reconstructed to be the last common ancestor of hemoglobins and myoglobins. Our findings reveal that AncMH harbors heme and that the heme binds oxygen. Furthermore, we demonstrate that the ferrous heme in AncMH is pentacoordinated, similar to that of human adult hemoglobin and horse myoglobin. A detailed comparison of the heme pocket structure indicates that the heme pocket in AncMH is more similar to that of hemoglobin than that of myoglobin. However, the autoxidation of AncMH is faster than that of both hemoglobin and myoglobin. Collectively, our results suggest that ancestral proteins of hemoglobins and myoglobins evolved in steps, including the hexa- to pentacoordination transition, followed by stabilization of the oxygen-bound form.

Chromophore Structure in an Inactive State of a Novel Photosensor Protein Opn5L1: Resonance Raman Evidence for the Formation of a Deprotonated Adduct at the 11th Carbon Atom.

Opsins are photosensitive G protein-coupled receptor proteins and are classified into visual and nonvisual receptors. Opn5L1 is a nonvisual opsin that binds all-trans retinal as a chromophore. A unique feature of Opn5L1 is that the protein exhibits a photocyclic reaction upon photoexcitation. Determining the chromophore structures of intermediates in the photocycle is essential for understanding the functional mechanism of Opn5L1. A previous study revealed that a long-lived intermediate in the photocycle cannot activate the G protein and forms a covalent bond between the retinal chromophore and a nearby cysteine residue. However, the position of this covalent bond in the chromophore remains undetermined. Here, we report a resonance Raman study on isotopically labeled samples in combination with density functional theory calculations and reveal that the 11th carbon atom of the chromophore of the intermediate forms a covalent linkage to the cysteine residue. Furthermore, vibrational assignments based on the isotopic substitutions and density functional theory calculations suggested that the Schiff base of the intermediate is deprotonated. The chromophore structure determined in the present study well explains the mechanism of the photocyclic reaction, which is crucial to the photobiological function of Opn5L1.

Protein dynamics of a light-driven Na+ pump rhodopsin probed using a tryptophan residue near the retinal chromophore.

Direct observation of protein structural changes during ion transport in ion pumps provides valuable insights into the mechanism of ion transport. In this study, we examined structural changes in the light-driven sodium ion (Na+) pump rhodopsin KR2 on the sub-millisecond time scale, corresponding with the uptake and release of Na+. We compared the ion-pumping activities and transient absorption spectra of WT and the W215F mutant, in which the Trp215 residue located near the retinal chromophore on the cytoplasmic side was replaced with a Phe residue. Our findings indicated that atomic contacts between the bulky side chain of Trp215 and the C20 methyl group of the retinal chromophore promote relaxation of the retinal chromophore from the 13-cis to the all-trans form. Since Trp215 is conserved in other ion-pumping rhodopsins, the present results suggest that this residue commonly acts as a mechanical transducer. In addition, we measured time-resolved ultraviolet resonance Raman (UVRR) spectra to show that the environment around Trp215 becomes less hydrophobic at 1 ms after photoirradiation and recovers to the unphotolyzed state with a time constant of around 10 ms. These time scales correspond to Na+ uptake and release, suggesting evolution of a transient ion channel at the cytoplasmic side for Na+ uptake, consistent with the alternating-access model of ion pumps. The time-resolved UVRR technique has potential for application to other ion-pumping rhodopsins and could provide further insights into the mechanism of ion transport.

Time-resolved spectroscopic mapping of vibrational energy flow in proteins: Understanding thermal diffusion at the nanoscale.

Vibrational energy exchange between various degrees of freedom is critical to barrier-crossing processes in proteins. Hemeproteins are well suited for studying vibrational energy exchange in proteins because the heme group is an efficient photothermal converter. The released energy by heme following photoexcitation shows migration in a protein moiety on a picosecond timescale, which is observed using time-resolved ultraviolet resonance Raman spectroscopy. The anti-Stokes ultraviolet resonance Raman intensity of a tryptophan residue is an excellent probe for the vibrational energy in proteins, allowing the mapping of energy flow with the spatial resolution of a single amino acid residue. This Perspective provides an overview of studies on vibrational energy flow in proteins, including future perspectives for both methodologies and applications.

Origin of a Double-Band Feature in the Ethylenic C═C Stretching Modes of the Retinal Chromophore in Heliorhodopsins.

Photoreceptor proteins play a critical role in light utilization for energy conversion and environmental sensing. Rhodopsin is a prototypical photoreceptor protein containing a retinal group that functions as a light-receptive site. It is essential to characterize the structure of the retinal chromophore because the chromophore structure, along with retinal-protein interactions, regulates which wavelengths of light are absorbed. Resonance Raman spectroscopy is a powerful tool to characterize chromophore structures in proteins. The resonance Raman spectra of heliorhodopsins, a recently discovered rhodopsin family, were previously reported to exhibit two intense ethylenic C═C stretching bands never observed for type-1 rhodopsins. Here, we show that the double-band feature in the ethylenic C═C stretching modes is not due to structural inhomogeneity but rather to the retinal polyene chain's linear structure. It contrasts with bent all-trans chromophore in type-1 rhodopsins. The linear structure of the chromophore results from weak atomic contacts between the 13-methyl group and a nearby Trp side chain, which can slow thermal reisomerization in the photocycle. It is possible that the deceleration of reisomerization increases the lifetime of the signaling intermediate for photosensory function.

Contact-Mediated Retinal-Opsin Coupling Enables Proton Pumping in Gloeobacter Rhodopsin.

When a chromophore embedded in a photoreceptive protein undergoes a reaction upon photoexcitation, the photoreaction triggers structural changes in the protein moiety that are necessary for the function of the protein. It is thus essential to elucidate the coupling between the chromophore and protein moiety to understand the functional mechanism for photoreceptive proteins, but the mechanism by which this coupling occurs remains poorly understood. Here, we show that nonbonded atomic contacts play an essential role in driving functionally important structural changes following photoisomerization of the chromophore in Gloeobacter rhodopsin (GR). Time-resolved ultraviolet resonance Raman spectroscopy revealed that the substitution of Trp222, which contacts with methyl groups of the retinal chromophore, with a Phe residue reduced the extent of structural change. The proton-pumping activity of the GR mutant was as small as 9% of that of the wild type. Time-resolved visible absorption and resonance Raman spectra showed that the photocycle of the mutant proceeded to the L intermediate following the all-trans to 13-cis photoisomerization step but did not result in the deprotonation of the chromophore. The present results demonstrate that the atomic contacts between the chromophore and the Trp222 side chain induce the structural changes necessary for proton transfer. The requirement for dense atomic packing in a protein structure for the efficient propagation of structural changes through a coupling mechanism is discussed.

Cis-Trans Reisomerization Precedes Reprotonation of the Retinal Chromophore in the Photocycle of Schizorhodopsin 4.

Recent discoveries of light-driven inward proton-pumping rhodopsins have opened new avenues to exploring the mechanism of unidirectional transport because these proteins transport protons in the opposite direction to conventional proton-pumping rhodopsins, despite their similar protein structure and membrane topology. Schizorhodopsin (SzR) is a newly discovered rhodopsin family of light-driven inward proton pumps. Here, we report time-resolved resonance Raman spectra showing that cis-trans thermal reisomerization precedes reprotonation at the Schiff base of the retinal chromophore in the photocycle of SzR AM_5_00977. This sequence has not been observed for the photocycles of conventional proton-pumping rhodopsins, in which reisomerization follows reprotonation, and thus provides insights into the mechanism of proton uptake to the chromophore during inward proton pumping. The present findings are expected to contribute to controlling the direction of proton transport in engineered proteins.

Dependence of Vibrational Energy Transfer on Distance in a Four-Helix Bundle Protein: Equidistant Increments with the Periodicity of α Helices.

Vibrational energy exchanges between various degrees of freedom are critical to barrier-crossing processes in proteins. Heme proteins are highly suitable for studies of the vibrational energy exchanges in proteins. The migration of excess energy released by heme in a protein moiety can be observed using time-resolved anti-Stokes ultraviolet resonance Raman spectroscopy. The anti-Stokes resonance Raman intensity of a tryptophan residue is an excellent probe for the excess energy and the spatial resolution of a single amino acid residue can be achieved. Here, we studied dependence of vibrational energy transfer on the distance in cytochrome b562, which is a heme-containing, four-helix bundle protein. The vibrational energy transfer from the heme group to a single tryptophan residue introduced by site-directed mutagenesis was examined for different heme-tryptophan distances by a quasi-constant length with the periodicity of α helices. Taken together with structural data obtained by molecular dynamics simulations, the energy transfer could be well described by the model of classical thermal diffusion, which suggests that continuum media provide a good approximation of the protein interior, of which the atomic packing density is very high.

High suitability of tryptophan residues as a spectroscopic thermometer for local temperature in proteins under nonequilibrium conditions.

Vibrational energy flow in the many degrees of freedom in proteins governs energy-barrier-crossing processes, such as conformational exchanges and thermal reactions. The intensity of anti-Stokes Raman bands arises from vibrationally excited populations and can thus function as a selective probe for the excess energy. Time-resolved observations of the anti-Stokes ultraviolet resonance Raman (UVRR) intensity of amino acid residues provide information about the flow of excess energy in proteins, with the spatial resolution of an amino acid residue. The answer to the question of whether the extent of vibrational excitation in any given vibrational modes reflects the extent of excitation in the whole molecule under nonequilibrium conditions is not straightforward. Here, we calculated the occupation probabilities of vibrational states for model compounds of amino acids under equilibrium and nonequilibrium conditions. At a given temperature, the occupation probability of the model compound of tryptophan under nonequilibrium conditions was nearly identical to that under equilibrium conditions at high temperature. Thus, the anti-Stokes band intensities of Trp residues in the nonequilibrium condition indicate the temperature of the molecules with equivalent energy in the equilibrium condition. In addition, we showed that the temperatures calculated on the basis of two UVRR bands of tryptophan in a time-resolved spectrum agreed with each other within the experimental uncertainty. The present results demonstrate that anti-Stokes UVRR bands of Trp residues serve as an excellent spectroscopic thermometer for determining the local temperature in proteins under nonequilibrium conditions.

Strongly Hydrogen-Bonded Schiff Base and Adjoining Polyene Twisting in the Retinal Chromophore of Schizorhodopsins.

A transmembrane proton gradient is generated and maintained by proton pumps in a cell. Metagenomics studies have recently identified a new category of rhodopsin intermediates between type-1 rhodopsins and heliorhodopsins, named schizorhodopsins (SzRs). SzRs are light-driven inward proton pumps. Comprehensive resonance Raman measurements were conducted to characterize the structure of the retinal chromophore in the unphotolyzed state of four SzRs. The spectra of all four SzRs show that the retinal chromophore is in the all-trans and 15-anti configuration and that the Schiff base is protonated. The polyene chain is planar in the center of the retinal chromophore and is twisted in the vicinity of the protonated Schiff base. The protonated Schiff base in the SzRs forms a stronger hydrogen bond than that in outward proton-pumping rhodopsins. We determined that the hydrogen-bonding partner of the protonated Schiff base is not a water molecule but an amino acid residue, presumably an Asp residue in helix G. The present observations provide valuable insights into the inward proton-pumping mechanism of SzRs.

Regulatory Switching by Concerted Motions on the Microsecond Time Scale of the Oxygen Sensor Protein FixL.

Signal transduction proteins perceive external stimuli in their sensor module and regulate the biological activities of the effector module, allowing cellular adaptation in response to environmental changes. FixL is a dimeric heme protein kinase that senses the oxygen level in plant root nodules to regulate the transcription of nitrogen fixation genes via the phosphorylation of its cognate transcriptional activator. Dissociation of oxygen from the heme induces conformational changes in the protein, converting it from the inactive form for phosphorylation to the active form. However, how FixL undergoes conformational change to regulate kinase activity upon oxygen dissociation remains poorly understood. Here we report time-resolved ultraviolet resonance Raman spectra showing conformational changes for FixL from Sinorhizobium meliloti. We observed spectral changes with a time constant of about 3 µs, which were oxygen-specific. Furthermore, we found that the conformational changes in the sensor and kinase domains are coupled, enabling allosteric control of kinase activity. Our results demonstrate that concerted structural changes on the microsecond time scale serve as the regulatory switch in FixL.

Resonance Raman Determination of Chromophore Structures of Heliorhodopsin Photointermediates.

Light is utilized as energy or information by rhodopsins (membrane proteins that contain a retinal chromophore). Heliorhodopsins (HeRs) are a new class of rhodopsins with low sequence identity (<15%) to microbial and animal rhodopsins. Their physiological roles remain unknown, although the involvement of a long-lived intermediate in the photocycle suggests a light-sensor function. Characterization of the molecular structures of the intermediates is essential to an understanding of the roles and mechanisms of HeRs. We determined the chromophore structures of the intermediates in HeR 48C12 by time-resolved resonance Raman spectroscopy and observed that the hydrogen bond of the protonated Schiff base strengthened prior to deprotonation. The chromophore is photoisomerized from the all-trans to the 13-cis form and is reisomerized in the transition from the O intermediate to the unphotolyzed state. Our results demonstrate that the chromophore structure evolves similarly to microbial rhodopsins, despite the dissimilarity in amino acid residues surrounding the chromophore.

Nonbonded Atomic Contacts Drive Ultrafast Helix Motions in Myoglobin.

The association and dissociation of small ligands regulate the functions of proteins through structural changes in the protein. Such structural changes propagate long distances, and this allostery plays a key role in molecular functions. However, the mechanism by which structural changes are transmitted is poorly understood. Here we show that nonbonded atomic contacts play an essential role in driving the displacement of a helix in picosecond time scale primary structural changes following the dissociation of carbon monoxide from the heme group in myoglobin. The present time-resolved ultraviolet resonance Raman study revealed that the amplitude of this helix displacement was reduced upon substitution of Val68, which contacts the heme in wild-type myoglobin, with a less bulky side chain (Ala). Our findings provided the first direct evidence that structural changes are transmitted not only by covalent bonds, salt bridges and hydrogen bonds but also by nonbonded atomic contacts in the primary protein response upon ligand dissociation. Furthermore, the present results indicate the importance of dense atomic packing in a protein structure for responding to the association and dissociation of small molecules. The high compactness of protein structures makes possible the propagation of structural changes, providing useful clues to the design of molecular machines.

Role of atomic contacts in vibrational energy transfer in myoglobin.

Heme proteins are ideal systems to investigate vibrational energy flow at the atomic level. Upon photoexcitation, a large amount of excess vibrational energy is selectively deposited on heme due to extremely fast internal conversion. This excess energy is redistributed to the surrounding protein moiety and then to water. Vibrational energy flow in myoglobin (Mb) was examined using picosecond time-resolved anti-Stokes ultraviolet resonance Raman (UVRR) spectroscopy. We used the Trp residue directly contacting the heme group as a selective probe for vibrationally excited populations. Trp residues were placed at different position close to the heme by site-directed mutagenesis. This technique allows us to monitor the excess energy on residue-to-residue basis. Anti-Stokes UVRR measurements for Mb mutants suggest that the dominant channel for energy transfer in Mb is the pathway through atomic contacts between heme and nearby amino acid residues as well as that between the protein and solvent water. It is found that energy flow through proteins is analogous to collisional exchange processes in solutions.

Allosteric Communication with the Retinal Chromophore upon Ion Binding in a Light-Driven Sodium Ion-Pumping Rhodopsin.

Krokinobacter rhodopsin 2 (KR2) serves as a light-driven sodium ion pump in the presence of sodium ion and works as a proton pump in the presence of larger monovalent cations such as potassium ion, rubidium ion, and cesium ion. Recent crystallographic studies revealed that KR2 forms a pentamer and possesses an ion binding site at the subunit interface. It is assumed that sodium ion bound at this binding site is not transported but contributes to the thermal stability. Because KR2 can convert its function in response to coexisting cation species, this ion binding site is likely to be involved in ion transport selectively. However, how sodium ion binding affects the structure of the retinal chromophore, which plays a crucial role in ion transport, remains poorly understood. Here, we observed the structure of the retinal chromophore under a wide range of cation concentrations using visible absorption and resonance Raman spectroscopy. We discovered that the hydrogen bond formed between the Schiff base of the retinal chromophore and its counterion, Asp116, is weakened upon binding of sodium ion. This allosteric communication between the Schiff base and the ion binding site at the subunit interface likely increases the apparent efficiency of sodium ion transport. In addition, this study demonstrates the significance of sodium ion binding: even though sodium ion is not transported, binding regulates the structure around the Schiff base and stabilizes the oligomeric structure.

Effect of a bound anion on the structure and dynamics of halorhodopsin from Natronomonas pharaonis.

Active ion transport across membranes is vital to maintaining the electrochemical gradients of ions in cells and is mediated by transmembrane proteins. Halorhodopsin (HR) functions as a light-driven inward pump for chloride ions. The protein contains all-trans-retinal bound to a specific lysine residue through a protonated Schiff base. Interaction between the bound chloride ion and the protonated Schiff base is crucial for ion transport because chloride ion movement is driven by the flipping of the protonated Schiff base upon photoisomerization. However, it remains unknown how this interaction evolves in the HR photocycle. Here, we addressed the effect of the bound anion on the structure and dynamics of HR from Natronomonas pharaonis in the early stage of the photocycle. Comparison of the chloride-bound, formate-bound, and anion-depleted forms provided insights into the interaction between the bound anion and the chromophore/protein moiety. In the unphotolyzed state, the bound anion affects the π-conjugation of the polyene chain and the hydrogen bond of the protonated Schiff base of the retinal chromophore. Picosecond time scale measurements showed that the band intensities of the W16 and W18 modes of the tryptophan residues decreased instantaneously upon photoexcitation of the formate-bound form. In contrast, these intensity decreases were delayed for the chloride-bound and anion-depleted forms. These observations suggest the stronger interactions of the bound formate ion with the retinal chromophore and the chromophore pocket. On the nanosecond to microsecond timescales, we found that the interaction between the protonated Schiff base and the bound ion is broken upon formation of the K intermediate and is recovered following translocation of the bound anion toward the protonated Schiff base in the L intermediate. Our results demonstrate that the hydrogen-bonding ability of the bound anion plays an essential role in the ion transport of light-driven anion pumps.

Distortion and a Strong Hydrogen Bond in the Retinal Chromophore Enable Sodium-Ion Transport by the Sodium-Ion Pump KR2.

We conducted a comprehensive time-resolved resonance Raman spectroscopy study of the structures of the retinal chromophore during the photocycle of the sodium-ion pump Krokinobacter rhodopsin 2 (KR2). We succeeded in determining the structure of the chromophore in the unphotolyzed state and in the K, L, M, and O intermediates, by overcoming the problem that only a small fraction of the M intermediate is accumulated in the KR2 photocycle. The Schiff base in the retinal chromophore forms a strong hydrogen bond in the unphotolyzed state and in the K, L, and O intermediates and is deprotonated in the M intermediate. Formation of this strong hydrogen bond facilitates deprotonation of the Schiff base, which is necessary for the sodium ion to move past the Schiff base. The polyene chain in the chromophore of KR2 is twisted in all of the states of the photocycle: the portion near the Schiff base is largely twisted in the unphotolyzed state and in the K intermediate, whereas the middle portion of the polyene chain becomes largely twisted in the L, M, and O intermediates. During the photocycle, the twisted structure of the polyene chain and strong hydrogen bond at the Schiff base are advantageous for transient relocation of the Schiff base proton. The obtained resonance Raman data clarified the unique structural features of the KR2 chromophore, which are not accessible by other methods.