Molecular properties of a viral heliorhodopsin, V2HeR2

Abstract

Heliorhodopsin (HeR) is a family of microbial rhodopsin discovered in 2018, whose genes are found from archaea, bacteria, unicellular eukaryotes, and giant viruses. Viral heliorhodopsins are classified into VHeR1–4 based on their amino acid sequences, and we previously reported the proton transport activity for V2HeR3. In this study, we report molecular properties of V2HeR2. V2HeR2 contains all-trans retinal predominantly in the dark, and the protonated Schiff base is stabilized by a counterion. The photocycle is described by the sequentially-formed K, M, and O intermediates. The O intermediate with a long lifetime (15.8 sec) and negligible ion transport activity implicate the light sensor function for V2HeR2, which is also the case for many HeRs. FTIR spectroscopy revealed that the chromophore structure is a distorted 13-cis form in the K and O intermediates. Although these properties are common for other HeRs, FTIR spectroscopy gain unique structural factors in the active O intermediate. The 13-cis chromophore is highly distorted near the Schiff base, and the hydrogen bond of the Schiff base is weaker than the resting state. The long-lived O intermediate with the distorted 13-cis retinal and without hydrogen bond of the Schiff base is unique in V2HeR2, which is regulated by the surrounding protein moiety. Strengthened hydrogen bond in amide-I band in the O intermediate of V2HeR2 is opposite to the case in Thermoplasmatales archaeon HeR (TaHeR) and HeR 48C12. Unique protein structural changes in V2HeR2, TaHeR, and HeR 48C12 are possibly correlated to different interaction with their partner proteins.

Molecular properties of V2HeR2, a viral heliorhodopsin, were studied by spectroscopic methods. The photocycle is described by the sequentially-formed K, M, and O intermediates like other HeRs. Long-lived O intermediate and lack of ion transport suggest light sensor function in V2HeR2. FTIR spectroscopy revealed unique protein structural changes in the O intermediate, including distorted 13-cis retinal and weak hydrogen bond of the Schiff base. Unlike other HeRs such as TaHeR and HeR 48C12, V2HeR2 shows strengthened hydrogen bond in the amide-I band, suggesting different interaction with their partner proteins.

Significance

V2HeR2, a viral heliorhodopsin, likely functions as a light sensor due to its long-lived O intermediate and lack of ion transport. FTIR spectroscopy revealed that unique protein structural changes in the O intermediate. The distorted 13-cis retinal without hydrogen bond of the Schiff base, which is regulated by the surrounding protein moiety. Unlike other HeRs such as TaHeR and HeR 48C12, V2HeR2 shows strengthened hydrogen bond in the amide-I band, suggesting different interaction with their partner proteins.

 Introduction

Rhodopsins are classified into type-1 microbial or type-2 animal rhodopsins, which contain all-trans or 11-cis retinal, respectively, as the chromophore [1–8]. Type-2 animal rhodopsins function as G-protein coupled receptors, while the functions of type-1 microbial rhodopsins are highly diverse and include light-driven ion pumps, light-gated ion channels, light sensors, and light-activated enzymes. Ion-transporting rhodopsins are used as the main tools in optogenetics [9–11]. In addition to type-1 and type-2 rhodopsins, a previously unrecognized diverse family, heliorhodopsins (HeRs), was discovered using functional metagenomics [12]. The most noticeable difference is that HeRs have inverted membrane topology compared to type-1 and -2 rhodopsins [12,13]. Extensive studies showed that structures and photocycles of HeRs resemble those of type-1 rhodopsins [14–41].

Similarly to type-1 rhodopsins, HeRs are encoded in genomes of archaea, bacteria, unicellular eukaryotes, and giant viruses. Although physiological functions of HeRs remained unknown, a proton-transporting HeR was discovered from a giant virus in 2022 [42]. In the same year, it was suggested that HeRs might interact with Glutamine synthase [43] and a photolyase repairing DNA damage [44] in a light-dependent manner. Protein-protein interaction inside membrane has recently been reported between ABC transporter and HeR [45]. It is thus likely that ion-transporting proteins are much smaller in population for HeRs than those in type-1 microbial rhodopsin, and the long photocycle of HeRs would be advantageous for activating interacting proteins.

The proton-transporting HeR, called V2HeR3, was found in giant double-stranded DNA viruses from the genus Coccolithovirus (Phycodnaviridae) that infect the microalga Emiliania huxleyi (=Gephyrocapsa huxleyi) [42]. E. huxleyi is a globally important marine coccolithophore whose massive blooms are observable from satellites and have an impact on Earth’s climate [46]. E. huxleyi viruses are able to collapse E. huxleyi blooms and thus represent one of the main factors controlling abundance of E. huxleyi in the ocean [47,48]. Coccolithoviruses such as EhV-201 and EhV-202 encode HeRs in their genomes. While EhV-201 has two HeR genes, V1HeR1 and V1HeR2, EhV-202 has three HeR genes, V2HeR1, V2HeR2 and V2HeR3 (Supplementary Figure S1). More than 3,000 HeRs can be classified into four groups (Figure 1), among which only one branch demonstrates light-dependent ion-transporting activity (a group of V2HeR3 in Figure 1) [12,13,42]. Therefore, when expressed in the E. huxleyi cell membranes, only V2HeR3 has the potential to depolarize the host cells by light, possibly to overcome the host defense mechanisms or to prevent superinfection [49].

Figure 1  A Phylogenetic tree of HeRs, which includes six viral HeRs from E. huxleyi virus 202 (V2HeR1–3), E. huxleyi virus 201 (V1HeR1, 2) and E. huxleyi virus PS401 (VPS401HeR), a eukaryotic HeR from E. huxleyi (EhHeR), an archaeal HeR (TaHeR) and a bacterial HeR (HeR48C12).

Here we studied the molecular properties of V2HeR2. We previously reported the λ_max (510 nm), photocycle time constant (16 sec) [13], and no light-dependent ion-transport activity [42]. In the present study, comprehensive analysis was performed for V2HeR2, including pH titration, HPLC analysis, photoreaction monitored at different wavelengths. In addition, light-induced difference FTIR spectra were measured at 100 and 293 K, which probe structural changes upon retinal photoisomerization and activation, respectively. Common and unique properties of V2HeR2 are discussed on the basis of the present spectroscopic observations.

 Materials and methods

 Phylogenetic reconstructions

An unrooted phylogenetic tree was constructed with MEGA12 software [50]. The protein sequences were aligned using MUSCLE. Evolutionary relationships of 33 taxa were inferred using the Neighbor-Joining method [51]. The optimal tree with the sum of branch length=4.395 is shown. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (500 replicates) are shown next to the branches [52]. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Poisson correction method [53] and are in the units of the number of amino acid substitutions per site. The analytical procedure encompassed 33 amino acid sequences. The pairwise deletion option was applied to all ambiguous positions for each sequence pair resulting in a final data set comprising 366 positions. Evolutionary analyses were conducted in MEGA12 utilizing up to 8 parallel computing threads. Sequence data of the heliorhodopsins were from the GenBank database. VHeR genes come from 14 EhVs [42].

 Protein expression and purification

The gene encoding N-terminal His-tagged V2HeR2 was cloned into the EcoRI and XbaI site of pPICZB vector (Thermo Fisher Scientific). The recombinant protein was expressed in the P. pastoris strain SMD1168H (Thermo Fisher Scientific) [54]. The cells were harvested 48–60 hr after expression was induced in BMMY medium when 10 mM of all-trans-retinal (Sigma-Aldrich) was supplemented in the culture to a final concentration of 30 μM. Additionally, 100% filtered methanol was added to the growth medium every 24 hr of induction to a final concentration of 0.5%. Membranes containing V2HeR2 was isolated as described elsewhere with the following modifications [55]. Washed P. pastoris cells were resuspended in buffer A (7 mM NaH₂PO₄, 7 mM EDTA, 7 mM DTT, and 1 mM phenylmethylsulfonyl fluoride [PMSF], pH 6.5) and slowly shaken with all-trans-retinal (added to a final concentration of 25 μM) in the dark at room temperature for 3–4 hr in the presence of 0.5% of Westase (Takara Bio, Inc) to digest the cell wall. The cells were disrupted by the two-time passage through a high-pressure homogenizer (EmulsiFlex C3, Avestin, Inc, Canada). The supernatants were centrifuged for 30 min at 40,000 rpm in a fixed-angle rotor, and the V2HeR2 membrane pellets were resuspended in solubilization buffer (20 mM KH₂PO₄, 1% n-dodecyl-β-D-maltoside (DDM), 1 mM PMSF, pH 7.5) and stirred overnight at 4°C. The solubilization mixture was centrifuged for 30 min at 40,000×g in a fixed-angle rotor. The solubilized protein was incubated with Ni-NTA agarose (QIAGEN, Hilden, Germany) for several hours. The resin with bound V2HeR2 was washed with wash buffer (50 mM KH₂PO₄, 400 mM NaCl, 0.1% DDM, 35 mM imidazole, pH 7.5) and then treated with elution buffer (50 mM KH₂PO₄, 400 mM NaCl, 0.1% DDM, 250 mM imidazole, pH 7.5). The collected fractions were dialyzed against a solution containing 50 mM KH₂PO₄, 400 mM NaCl, 0.1% DDM at pH 7.5 to remove the imidazole.

 Measurement of absorption spectra and pH titration

Absorption spectra of V2HeR2 were measured with a UV-visible spectrometer (V-2400PC, Shimadzu). To investigate the pH dependence, a solution containing 6 μM protein was solubilized in a six-mix buffer (10 mM citrate, 10 mM MES, 10 mM HEPES, 10 mM MOPS, 10 mM CHES and 10 mM CAPS). pH was changed at approximately 0.5 intervals by adding either concentrated HCl or NaOH.

 HPLC analysis of retinal configuration

The HPLC was equipped with a silica column (6.0×150 mm; YMC-Pack SIL, YMC, Japan), a pump (PU-2080, JASCO, Japan), and a UV-visible detector (UV-2070, JASCO) [56]. The solvent was composed of 12% (v/v) ethyl acetate and 0.12% (v/v) ethanol in hexane with a flow rate of 1.0 ml/min. Retinal oxime was formed by a hydrolysis reaction with the sample in 100 μl solution at 0.1 mg/ml protein concentration and 50 μl hydroxylamine solution at 1 M at 0°C. To ensure all the protein molecules reacted completely, 300 μl of methanol was added to denature the proteins. For light-adapted V2HeR2, the sample solution was illuminated with 540±10 nm light (interference filter, Toshiba) for 1 min before denaturation and extraction. Then, the retinal oxime was immediately extracted using hexane and 300 μl of solution was injected into the HPLC system. The molar composition of the retinal isomers was calculated from the areas of the corresponding peaks in the HPLC patterns. The assignment of the peaks was performed by comparing them with the HPLC pattern from retinal oximes of authentic all-trans, 13-cis, and 11-cis retinals. Independent measurements were repeated 2 or 3 times.

 Laser flash photolysis

For the laser flash photolysis measurement, V2HeR2 was purified and solubilized in 0.1% DDM, 400 mM NaCl, and 50 mM KH₂PO₄ (pH 7.5). The absorption of the protein solution was adjusted to 0.5 (total protein concentration ~0.25 mg/ml) at λ_max=519 nm. The sample was illuminated with a beam from a flash of second harmonics of a nanosecond-pulsed Nd-YAG laser (λ=532 nm, INDI40, Spectra-Physics). The laser energy density was ∼2.5 mJ/cm² per pulse, and the repetition period was set sufficiently slower than the photocycle rate of V2HeR2 to avoid unnecessary photoexcitation of the transient intermediates [13]. The output of an Xe arc lamp (L9289-01, Hamamatsu Photonics, Japan), was passed through a monochromator (S-10, SOMA OPTICS, Japan) to prepare a probe light at a specific wavelength. The time evolution of flash-induced absorbance change was obtained by measuring the transmitted intensity of the probe light after photoexcitation by a photomultiplier tube (R10699, Hamamatsu Photonics, Japan). The signal from the photomultiplier tube was averaged and stored using a digital-storage-oscilloscope (DPO7104, Tektronix, Japan). The sets of time-resolved transient absorption data were analyzed using Igor Pro ver. 9 and the Global Fit package with multiple exponential functions to obtain the decay time constants and pre-exponential factors.

 Light-induced difference transmission FTIR spectroscopy at 100 K

The purified proteins of V2HeR2 were reconstituted into a mixture of asolectin membranes with a protein-to-lipid molar ratio of 1:20 by removing DDM using Bio-Beads (SM-2; Bio-Rad, CA). The reconstituted samples were washed three times with 1 mM NaCl and 2 mM KH₂PO₄ (pH 7.5). The pellet was resuspended in the same buffer, where the concentration was adjusted to make the intensity of amide I~0.7. A 60 μl aliquot was placed onto a BaF₂ window and dried gently at 4°C. The films were then rehydrated with 1 μl H₂O or D₂O, and allowed to stand at room temperature for a few minutes to complete the hydration. For UV-visible spectroscopy, the sample film was hydrated with H₂O, and placed and cooled in an Optistat DN cryostat (Oxford Instruments, Abingdon, UK) mounted in a UV-vis spectrometer (V-550, JASCO, Japan) [57]. For FTIR spectroscopy, the sample film was hydrated with H₂O or D₂O, and placed and cooled in an Oxford Optistat DN2 cryostat mounted in a Cary670 spectrometer (Agilent Technologies, Japan) [13].

For the formation of the K intermediate, samples of V2HeR2 were illuminated with 520±10 nm light (interference filter, Toshiba) from a 1 kW tungsten-halogen projector lamp (Rikagaku) for 1 min at 100 K. The K intermediate was photo-reversed with λ>570 nm light (O-58 cut-off filter, AGC Techno Glass) for 2 min, followed by illumination with 520 nm light. The 128 interferograms were accumulated with 2 cm^–1 spectral resolution for each measurement. To increase the S/N ratio in FTIR spectroscopy, photoconversions to the K intermediate at 100 K was repeated 38 times in H₂O and 40 times in D₂O, respectively.

 Light-induced difference ATR FTIR spectroscopy at 293 K

Light-induced structural changes in the O intermediate of V2HeR2 were measured by attenuated total reflection FTIR (ATR–FTIR) spectroscopy. ATR–FTIR spectra were recorded in kinetics mode at 2 cm^–1 resolution range of 4000–700 cm^–1, using an FTIR spectrometer (Agilent) equipped with a liquid nitrogen-cooled mercury-cadmium-telluride (MCT) detector. In the measurements, the lipid-reconstituted V2HeR2 sample was placed on the surface of a silicon ATR crystal (Smiths, three internal total reflections) and dried naturally. After confirming that the value of the amide I band (1650 cm^–1), which is the standard for the absolute amount of protein, is 0.7, sample was rehydrated with 5 mL 150 mM NaCl and 10 mM HEPES-Na (pH 7.0). The film sample was rehydrated using a glass cell and maintained at 293 K using a temperature controller. The light irradiating conditions of V2HeR2 were such that O intermediate was accumulated by irradiating with 520±10 nm light (interference filter, Toshiba) for 5 minutes, and then returned to the initial state by keeping dark condition for 5 minutes. The last 3 minutes spectra were used for the light-induced difference spectra.

 Results

 Molecular properties of the resting state in V2HeR2

Figure 2 shows the absorption spectra of V2HeR2 in detergent (0.1% DDM) at pH 7.5. The absorption spectrum of V2HeR2 possesses the λ_max at 519 nm, which is 19 nm red-shifted from V2HeR3 (500 nm) [42] and ~30 nm blue-shifted from many HeRs such as 48C12 and TaHeR [12,13].

Figure 2  UV-visible absorption spectrum of V2HeR2, which were solubilized in 0.1% DDM at pH 7.5.

To further study the molecular properties of V2HeR2, we measured absorption spectra at different pHs. Figure 3a shows the absorption spectra of V2HeR2 at low pH. When pH is decreased from pH 7.28, a spectral red-shift was observed. This is commonly reported for many microbial rhodopsins and reflects the protonation of counterions. At pH 0.7, the λ_max is shifted to ~430 nm, presumably owing to the acid denaturation of the protein. The pKa of E113 in V2HeR2 was determined to be 3.4 (Figure 3b), which was lower than that of V2HeR3 (4.3) [42], but close to those of 48C12 (3.7) [12], and TaHeR (3.6) [13]. Figure 3c shows the difference absorption spectra of V2HeR2 at high pH. When pH is increased, the absorption in rhodopsins is shifted to <400 nm by the deprotonation of the Schiff base. This was similarly observed for V2HeR2 (Figure 3c), and the pKa was determined to be 12.9 (Figure 3d). This value was lower than that of V2HeR3 (14.9) [42], but higher than those of 48C12 (11.5) [12], and TaHeR (11.2) [13].

Figure 3  UV-visible absorption of heliorhodopsin V2HeR2 at different pH values. (a) (b) Red-shift of UV-visible absorption spectrum of V2HeR2, and protonation of the counterion. UV-visible absorption spectra (a) and the λ_max (orange circles) of V2HeR2 at pH 0.70–7.28 (b). (c) (d) Deprotonation of the retinal Schiff base of V2HeR2 at alkaline pH. Difference absorption spectra (c) and absorption change at λ=520 nm (blue circles) of V2HeR2 upon pH change from 8.68 to higher values. pKa value indicates mean±s.d., which is fitted with the Henderson–Hasselbalch equation (dashed line). This experiment was performed once.

Figure 4 shows HPLC pattern of chromophores extracted from V2HeR2. Most of the retinal (95%) bound to V2HeR2 adopts an all-trans configuration in the dark. When the retinal was extracted after illumination, the proportion of the 13-cis form increased to 25%. This property is essentially similar to other HeRs and type-1 rhodopsins.

Figure 4  HPLC pattern of retinal extracted from V2HeR2 in the dark (black line) and under illumination at λ=540±10 nm (red line). Most of the retinal (95%) bound to V2HeR2 adopts an all-trans configuration in the dark (n=3). When the retinal is extracted after illumination, the proportion of the 13-cis form increased to 25% (n=2).

 Photoreaction dynamics of V2HeR2

We next studied the photoreaction dynamics of V2HeR2 after laser excitation at 532 nm. Figure 5a shows the transient absorption result of V2HeR2 at pH 7.5, where absorbances were monitored at the absorption wavelengths of V2HeR2 (520 nm), the M intermediate (400 nm), and the K/O intermediates (570 nm). Negative signal at 520 nm corresponds to the bleaching of the resting state by light, and the recovery was slow (time constant of 15.8 sec). Positive signal at 570 nm at wide time scale shows that red-shifted intermediates such as K and O dominate the photocycle of V2HeR2. Rise signal at ~10^–3 sec indicates the presence of two late (O) intermediates, and the transition from the O₁ to O₂ state occurs with a time constant of 3.3 msec. Small positive signal at 400 nm for 10^–5 to 10^–4 sec shows formation of the M intermediate, and global fitting analysis of three wavelengths (solid lines in Figure 5a) resulted in the rise and decay time of the M intermediate to be 20 μsec and 109 μsec, respectively. Photocycle dynamics of V2HeR2 are summarized in Figure 5b.

Figure 5  Photocycle of V2HeR2. (a) Time evolutions of transient absorption change at specific wavelengths. Samples were excited at 532 nm. Each of the wavelengths probes different photochemical species: 400 nm for the M intermediate; 520 nm for the bleached dark state; and 570 nm for the K and O intermediates. (b) Photoreaction schema of V2HeR2, determined by the multi-exponential fitting for the time evolution of the transient absorption changes shown in (a).

Between V2HeR2 and V2HeR3, the photocycle is much slower for the former (15.8 sec) than for the latter (59% 0.41 sec and 41% 3.4 sec) [42]. While V2HeR3 transports protons, V2HeR2 does not have ion-transporting activity, suggesting that V2HeR2 activates other proteins and the long photocycle would be advantageous for this aim. Recently, Nakamura et al. revealed the kinetic determinant as an interhelical hydrogen bond between trans membrane 3 (TM3) and trans membrane 4 (TM4) [40]. HeR 48C12 with Ser in TM3 and Asn in TM4 (named SNap bond) exhibits 14-times faster photocycle than TaHeR with Ala in TM3 and Thr in TM4 (Figure S1), and interconverting mutants of 48C12 and TaHeR reversed each kinetics. Considering that corresponding residues in VeHeR2 are Ala in TM3 and Ile in TM4, the absence of the hydrogen bond is also the case for VeHeR2 and the slow photocycle is consistent with that of TaHeR. However, it should be noted that the photocycle of V2HeR3 is rather fast despite that it possesses Ala in TM3 and Thr in TM4 like TaHeR. The M intermediate is dominated in the photocycle of V2HeR3 [42] for proton transport, where a different mechanism presumably works to control the kinetics.

 Low-temperature light-induced difference FTIR spectroscopy of V2HeR2 at 100 K

Figure 6 compares light-minus-dark difference FTIR spectra of V2HeR2 in H₂O (black line) and D₂O (red line) at 100 K (top panel), which were compared with those of V2HeR3 (middle panel) [42] and TaHeR (bottom panel) [13]. The low frequency shift of the C=C stretch from 1551/1540 cm^–1 in V2HeR2 to 1546/1531 cm^–1 is consistent with the spectral red-shift upon formation of the K intermediate, which were also the case for V2HeR3 and TaHeR. Presence of two peaks suggests heterogeneous chromophore structure in the resting state of V2HeR2. The peak pair at 1199 (–)/1180 (+) cm^–1 in V2HeR2 (top panel in Figure 6) is characteristic of the difference spectra upon all-trans to 13-cis photoisomerization, which were similarly observed for V2HeR3 and TaHeR. Interestingly, V2HeR2 exhibits two negative peaks at 1199 and 1192 cm^–1 in D₂O, also suggesting heterogeneous chromophore structure of the all-trans form in the resting state. Lack of the negative 1192-cm^–1 band in H₂O is unclear, but another positive band at this frequency may cancel it.

Figure 6  Light-induced difference FTIR spectra of V2HeR2 at 100 K (top panel), V2HeR3 at 100 K (middle panel) and TaHeR at 77 K (bottom panel) in the 1800–800 cm^–1 region. The sample was reconstituted into lipid membranes, and membrane films are hydrated with H₂O (black line) and D₂O (red line). The spectra of V2HeR3 and TaHeR are reproduced from [42] and [13], respectively.

The 1000-900 cm^–1 region is characteristic of hydrogen out-of-plane (HOOP) vibrations, which are informative of chromophore distortion. Top panel of Figure 6 shows complex spectral feature at these frequencies. Peaks are observed at 1010 (+)/966 (–)/961 (+)/957 (–)/950 (+) cm^–1 in H₂O, and at 1007 (–)/988 (–)/980 (+)/966 (–)/961 (+)/957 (–)/950 (+) cm^–1 in D₂O. D₂O-specific negative peak at 1007 and 988 cm^–1 is possibly ascribable to the in-plane N-D bending vibration of the Schiff base. A positive peak at 1010 cm^–1 in H₂O is down-shifted to 980 cm^–1 in D₂O, which originates from the HOOP vibration near the Schiff base, such as C15-HOOP. H/D unexchangeable peaks at 966 (–)/961 (+)/957 (–)/950 (+) cm^–1 come from HOOP in the central moiety of the retinal chromophore. Similar HOOP modes were observed for V2HeR3 (968 (–)/964 (+) cm^–1) and TaHeR (964 (–)/958 (+) cm^–1).

Positive peaks of V2HeR2 at 1404, 1317, and 1119 cm^–1 are up-shifted to 1417, 1323, and 1132 cm^–1, respectively, in D₂O (top panel of Figure 6). This is unusual as deuteration increases mass, leading to low frequency shift in general. Exceptional examples can be seen when vibrations are coupled. As these three peaks are strong, the retinal chromophore would contribute, such as N-H (N-D) bending vibrations. However, such an H/D effect was unclear from recent time-resolved resonance Raman spectroscopy [22]. Therefore, we infer these vibrations owing to protein at present. Similar peaks were observed for V2HeR3; positive peaks at 1403, 1318, and 1125 cm^–1 in H₂O are up-shifted to 1419, 1324, and 1138 cm^–1, respectively, in D₂O (middle panel of Figure 6), but not for TaHeR. Therefore, these vibrations are possibly unique for viral HeRs. Further study by use of isotope is intriguing.

The 1800-1600 cm^–1 region contains various protein vibrations including amide-I, the C=O stretch of peptide backbone. The C=N stretching vibration of the protonated Schiff base only appears as the chromophore vibration in this region. The C=N stretching mode is coupled to the N-H bending vibration of the Schiff base, by which C=NH stretch is up-shifted from C=ND stretch. The difference in frequency between H₂O and D₂O has been regarded as a measure of hydrogen-bonding strength of the Schiff base [58]. In case of V2HeR2, the negative peaks at 1657 cm^–1 in H₂O and at 1641 cm^–1 in D₂O are candidates of the C=NH and C=ND stretches, respectively (top panel of Figure 6). The difference (16 cm^–1) is smaller than those of V2HeR3 (21 cm^–1) (middle panel) and TaHeR (25 cm^–1) (bottom panel), suggesting that the hydrogen bond of the Schiff base in V2HeR2 is weaker than in V2HeR3 and TaHeR. The positive peaks at 1631 cm^–1 in H₂O and at 1611 cm^–1 in D₂O (top panel of Figure 6) are candidates of the C=NH and C=ND stretches in the K intermediate of V2HeR2, respectively. Therefore, hydrogen-bonding strength of the Schiff base is similar before and after retinal isomerization in V2HeR2.

The 1800-1700 cm^–1 region is characteristic of C=O stretches of protonated carboxylic acids. No clear bands were observed for V2HeR2 (top panel of Figure 6) as well as TaHeR (bottom panel of Figure 6). In contrast, V2HeR3 exhibits the bands at 1721 (–)/1712 (+) cm^–1, which is shifted to 1718 (–)/1704 (+) cm^–1 in D₂O (middle panel of Figure 6). This observation shows that a protonated carboxylic acid forms stronger hydrogen bond upon retinal photoisomerization in V2HeR3, but not in V2HeR2 and TaHeR. E191 is a unique residue in V2HeR3 and prerequisite for the proton transport activity [42], which is a candidate of the bands. Such carboxylic acids are absent in V2HeR2 like TaHeR.

 Room-temperature light-induced difference FTIR spectroscopy of V2HeR2

If V2HeR2 interacts with other proteins in a light-dependent manner, late intermediates such as O are the active state. Therefore, we next measured difference FTIR spectra of late intermediates of V2HeR2. As the photocycle of V2HeR2 is long, we illuminate the sample attached onto ATR cells in buffer, and difference FTIR spectra were obtained by subtracting the dark state spectrum from the spectrum recorded during illumination by means of ATR-FTIR spectroscopy at 293 K. The O intermediate was accumulated during illumination. We applied the same method to TaHeR [13], which is shown in Figure 7 (bottom panel). The photocycle of V2HeR3 was not enough long for this method, and the difference FTIR spectra of the after illuminatoin-minus-dark state were measured for a hydrated film by means of transmission FTIR spectroscopy at 240 K in the previous report [42].

Figure 7  Light-induced difference FTIR spectra of V2HeR2 at 293 K (top panel)), V2HeR3 at 240 K (middle panel) and TaHeR at 293 K (bottom panel)) in the 1800–800 cm^–1 region. The spectra of V2HeR3 and TaHeR are reproduced from [42]. Note that the spectra of V2HeR2 and TaHeR were obtained for membrane films in buffer by ATR-FTIR spectroscopy, while those of V2HeR3 were obtained for hydrated films by low-temperature transmission FTIR spectroscopy.

The low frequency shift of the C=C stretch from 1555/1537 cm^–1 to 1519 cm^–1 is consistent with the spectral red-shift upon formation of the O intermediate in V2HeR2, which was also the case for V2HeR3 and TaHeR. Two negative peaks for the difference with the K intermediate (Figure 6) are also observed for the difference with the O intermediate. The peak pair at 1197 (–)/1180 (+) cm^–1 in V2HeR2 (top panel in Figure 7) is also characteristic of the difference spectra upon all-trans to 13-cis photoisomerization, indicating that the O intermediate contains the 13-cis retinal as the chromophore, as well as 48C12 and TaHeR.

Strong bands in the HOOP region (1000-900 cm^–1) is characteristic for primary intermediates, which normally decreased in late intermediates by relaxation of the chromophore distortion. Nevertheless, top panel of Figure 7 shows strong HOOP vibration at 991 cm^–1 H₂O and at 975 cm^–1 D₂O, whose amplitudes are about half of C=C (1519 cm^–1) and C-C (1180 cm^–1) stretches. This indicates that the O intermediate contains a distorted 13-cis chromophore in V2HeR2. Similar observations were reported for HeR 48C12 by means of time-resolved resonance Raman spectroscopy [22]. In addition to the H/D exchangeable HOOP band, small peak pair was observed at 965 (–)/962 cm^–1 both in H₂O and D₂O (top panel of Figure 7). As top panel of Figure 6 shows 966 (–)/961 (+)/957 (–)/950 (+) cm^–1, this observation suggests that the chromophore distortions described by the 966 (–)/961 (+) cm^–1 and 957 (–)/950 (+) cm^–1 bands upon retinal photoisomerization (K intermediate) are retained and relaxed in the O intermediate, respectively.

Positive bands at 1631 cm^–1 H₂O and at 1623 cm^–1 D₂O are ascribable to the C=NH and C=ND stretches in the O intermediate of V2HeR2, respectively. The differences in frequency between H₂O and D₂O are 16 cm^–1 in V2HeR2, 20 cm^–1 in the K intermediate, and 8 cm^–1 in the O intermediate. Therefore, hydrogen-bond of the Schiff base is strengthens upon retinal photoisomerization in V2HeR2, but becomes weak in the O intermediate, which is weaker than the resting state. We infer that specific chromophore distortion near the Schiff base accompanies broken hydrogen bond of the Schiff base.

Three peaks at 1119, 1317, 1404 cm^–1 exhibiting up-shift in D₂O are unique to the K intermediate of V2HeR2, whereas such bands were unclear for the O intermediate (top panel of Figure 7). Therefore, such bands are specific to the primary intermediate, and relax in the O intermediate.

Activation of rhodopsins accompanies their large structural changes, which are well reflected by spectral changes in amide-I region. Amide-I vibration corresponds to C=O stretches of peptide backbone, and are highly sensitive to secondary structural alterations such as helices and sheets. The largest peak pair appears in the 1200-1180 cm^–1 region, C-C stretch of the retinal chromophore, at 100 K (top panel in Figure 6), but the largest peak pair appears in the 1670-1630 cm^–1 region, amide-I bands, at 293 K (top panel in Figure 7). This observation corresponds to large secondary structural alterations in the O intermediate of V2HeR2.

In case of V2HeR2, spectral downshift from 1671 and 1663 cm^–1 to 1647 and 1631 cm^–1 was observed in the amide-I region (top panel in Figure 7), indicating that hydrogen bond of peptide backbone is strengthened in the O intermediate. This is in contrast to TaHeR that exhibits spectral upshift from 1666 and 1655 cm^–1 to 1673 cm^–1 in the amide-I region (bottom panel in Figure 7). Spectral changes are more complex in V2HeR3 (middle panel in Figure 7). Both V2HeR2 and TaHeR presumably interact with other proteins in the O intermediate, and different peptide backbone signals may reflect unique modes of structural changes.

Additionally, the characteristic band at 1703 cm^–1 was observed in V2HeR2 (top panel in Figure 7). Since this band does not shift in D₂O, the C=O stretch of lipids is a possible candidate. Formation of the active O intermediate possibly changes structures of lipids by large secondary structural changes of V2HeR2. The 1671 cm^–1 in V2HeR2 likely corresponds to a structural change in the α_II helix (top panel in Figure 7), as well as the case (1666 cm^–1) in TaHeR (bottom panel in Figure 7). α_II helix has a slightly deformed helical structure which caused by additional hydrogen bonds from side chains of the Ser and Thr residues. The time-resolved dual-comb quantum cascade laser (QCL) spectra in 48C12, which contains an SNap-bond, revealed higher ratio for the absorption change of α_II helix to that of α_I helix [40]. In V2HeR2, lower ratio for the absorption change of α_II helix (1671 cm^–1) to that of α_I helix (1663 cm^–1) is consistent with the absence of an SNap-bond in V2HeR2.

 Discussion

Since the discovery of heliorhodopsins in 2018 [12], the function of V2HeR3 was first identified as proton transport in 2022 [42]. Nevertheless, most HeRs do not have ion transport activity, and sensor functions have been taken into account. In fact, protein-protein interactions of HeRs with glutamine synthase [43], a photolyase [44], and ABC transporter [45] were reported. In these cases, photoreaction cycles are almost characterized by a long-lived O intermediate [44,45]. While no interacting proteins have been identified for TaHeR or 48C12, similar photoreaction cycle characteristics were observed [12,13]. The retinal isomerization in TaHeR or 48C12 follows the typical pattern for type-1 microbial rhodopsins, where retinal configuration is all-trans form in the resting state and changed to the 13-cis form in the active O intermediate [12,13]. In contrast, the photoreaction cycle of V2HeR3 with proton transport activity, is characterized by a long-lived M intermediate [42]. Additionally, V2HeR3 contains both all-trans and 13-cis retinals in the resting state [42]. Therefore, molecular properties are likely distinct between HeRs with and without ion-transport.

This study reports the basic molecular properties of V2HeR2. V2HeR2 is similar with TaHeR in its photoreaction cycle and retinal isomerization, but FTIR results reveal distinct structural changes. From the measurement at 100 K, structural heterogeneity in the retinal chromophore of V2HeR2 was suggested (Figure 6). Chromophore distortions in the K intermediate resemble among V2HeR2, V2HeR3, and TaHeR with some modifications. Hydrogen-bonding strength of the retinal Schiff base is weaker in V2HeR2 than in V2HeR3 and TaHeR. Fully unexpected observation for the K intermediate was the three peaks at 1400-1100 cm^–1 exhibiting up-shift in D₂O (Figure 6). As they were unclear for the O intermediate (Figure 7), these bands are unique to the primary intermediate, and relax in the O intermediate. N-H (N-D) bending vibrations of the retinal Schiff base are candidates, but such an H/D effect was unclear from recent time-resolved resonance Raman spectroscopy [22]. Similar bands were observed for V2HeR3, but not for TaHeR, suggesting it is specific to viral HeRs. If these bands originate from protein, their origins are intriguing.

The M intermediate appears with a time constant of 20 μs in V2HeR2, but the population was small in transient absorption (Figure 5), so that we could not measure the difference FTIR spectra of the M intermediate. Formation of the M intermediate accompanies deprotonation of the Schiff base. Although the proton acceptor has been unclear, the water cluster near E108 was suggested from its mutation study [36]. Despite no structural information, water-containing hydrogen-bonding network probably exists in V2HeR2 as well as other HeRs [13,18,20,28], and transient proton transfer reactions lead to the active O intermediate.

Present transient absorption study detected two O states, O₁ and O₂, while the active state is probably the O₂ intermediate that appear with a time constant of 3.3 ms (Figure 5). Present FTIR study showed that the O intermediate (O₂ intermediate) contains the 13-cis retinal as the chromophore, as well as 48C12 and TaHeR [12,13]. Chromophore distortion near the Schiff base is characteristic of the O intermediate in V2HeR2. The hydrogen-bond of the Schiff base, strengthens upon retinal photoisomerization, becomes weak in the O intermediate, which is weaker than the resting state in V2HeR2. Therefore, the long-lived O intermediate is characterized by the distorted 13-cis retinal without hydrogen bond of the Schiff base. Recent time-resolved resonance Raman spectroscopy of HeR 48C12 showed similar hydrogen-bonding strength between the resting state and the O intermediate [22]. The unique chromophore structure in the O intermediate of V2HeR2 is the result of the surrounding proteins, and strengthened hydrogen bond in amide-I band in the O intermediate is characteristic of V2HeR2, which is opposite to the case in TaHeR (Figure 7) [13], and in HeR 48C12 [12]. Unique protein structural changes in V2HeR2, TaHeR, and HeR 48C12 are possibly correlated with different interaction with their partner proteins. Light-induced unfolding of helical structure in the O intermediate was suggested by transient grating (TG) and circular dichroism (CD) spectroscopy of TaHeR [34], which presumably enables protein-protein interaction.

Recent spectroscopic study revealed the determinant of the rise/decay of the O intermediate to be an SNap-bond, an interhelical hydrogen bond between Ser in TM3 and Asn in TM4 [40]. HeR 48C12 contains the SNap-bond, whose photocycle is faster than that of TaHeR. VeHeR2 contains Ala in TM3 and Ile in TM4, being consistent with the slow photocycle like TaHeR (Figure S1). While amide-I band of α helix (α_I helix) is observed at 1660-1650 cm^–1, slightly deformed helical structure exhibits amide-I band at 1670-1660 cm^–1, and called α_II helix, which is caused by additional hydrogen bonds from side chains of the Ser and Thr residues. The time-resolved dual-comb QCL spectra in 48C12 revealed higher ratio for the absorption change of α_II helix to that of α_I helix [40]. V2HeR2 exhibits a strong negative peak at 1663 cm^–1 with a shoulder at 1671 cm^–1 (top panel in Figure 7). Because of the presence of a sharp positive peak at 1647 cm^–1, negative amide-I band of the normal α helix (α_I helix) probably appears at 1663 cm^–1 in V2HeR2, and the negative bands at 1671 cm^–1 and 1663 cm^–1 correspond to α_II and α_I helix, respectively (top panel in Figure 7). Lower ratio for the absorption change of α_II helix (1671 cm^–1) to that of α_I helix (1663 cm^–1) in V2HeR2 is consistent with the absence of an SNap-bond.

 Conclusion

Viral heliorhodopsins are classified into VHeR1–4 based on their amino acid sequences, where VHeR3 and VHeR4 exhibit proton transport activity. The present study revealed the molecular characteristics of V2HeR2, belonging to VHeR2. Interaction with other proteins via its long-lived O intermediate was suggested, whose structural changes differ from those in TaHeR and HeR 48C12. EhV-202 encodes VHeR1–3, and we now know significant functional differences for V2HeR2 and V2HeR3. It is possible that V2HeR2 plays a role in viral survival strategies during host infection. Amino acid sequence of VHeR1 is also unique, containing a high amount of Pro at the C-terminus (Figure S1). Highly conserved two His, H23 and H87 in V2HeR2, are substituted with Asn (N22 and N84) in V2HeR1, suggesting different molecular properties from those of VHeR2 and VHeR3. Further study of V2HeR1 will lead to full understanding of the roles of HeRs in EhV-202.

 Conflict of interest

All authors declare that they have no conflicts of interest.

 Author contributions

H. K. directed the research and wrote the manuscript. R. M. prepared samples and performed all experiments and analysis with the help of K. K., M. K., K. I., and O. B. All authors discussed and commented on the manuscript.

 Data availability

The evidence data generated and/or analyzed during the current study are available from the corresponding author on reasonable request.

 Acknowledgements

This work was financially supported by grants from the Japanese Ministry of Education, Culture, Sports, Science and Technology to K.K. (18K14662) and H.K. (18H03986, 19H04959, 21H04969), and from Japan Science and Technology Agency (JST), PRESTO to K.K. (JPMJPR19G4) and CREST to H.K. (JPMJCR1753), and by the Israel Science Foundation (grant 1207/24 to O.B.). O.B. holds the Louis and Lyra Richmond Chair in Life Sciences.

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