Microbial rhodopsins are photoreceptive membrane proteins of microorganisms with diverse functions, light-driven ion pumps, light-gated ion channels, phototaxis sensors, light-dependent enzymes, and so on.1, 2, 3 Most microbial rhodopsins consist of seven transmembrane helices (TM1–7) and an all-trans-retinylidene Schiff base covalently bonded to a conserved lysine residue in TM7, which is the so-called retinal chromophore.1, 3 The characteristic motif of three amino acid residues in TM3 are tightly connected to the function of the rhodopsins. In bacteriorhodopsin (BR), which is the best-characterized outward proton-pumping rhodopsin, the motif residues are Asp85, Thr89, and Asp96 (DTD-motif). Asp85 and Asp96 work as proton acceptor and donor for the retinal Schiff-base (RSB), respectively (Figure 1(A)).1, 3

A new rhodopsin with a Thr–Ala–Thr (TAT) motif was recently found in the genome of the cultured marine heterotrophic alphaproteobacterium HIMB114 from the abundant SAR11 clade (Pelagibacterales).4, 5 This rhodopsin, called TAT rhodopsin, could be heterologously expressed in Escherichia coli cells, and the expressed protein was colored by binding the retinal chromophore.6 As no ion transport was observed upon light illumination, TAT rhodopsin was considered to have a function other than being an ion pump or channel, but it remains unclear so far. On the other hand, the purified TAT rhodopsin shows unique spectroscopic characteristics.6 In contrast to other microbial rhodopsins, that have an absorption peak in the visible region, TAT rhodopsin exhibits two absorption peaks in the UV (∼400 nm) and visible (∼560 nm) regions.6 While the RSB is protonated in the visible-absorbing state as other microbial rhodopsins, the UV-absorbing state has a deprotonated retinal chromophore. In general, visible photon absorption induces all-trans-to-13-cis isomerization of the retinal chromophore in most microbial rhodopsins1, 2, 3 leading to the sequential transformation between subsequent photointermediates that are distinguished by different UV–visible absorption spectra. These transformations eventually return to the initial state thus completing the so-called photocycle reaction. In contrast, the photoexcitation of the visible-absorbing state of TAT rhodopsin gives rise to the first red-shifted state, the so-called K intermediate, that quickly returns to the initial state with 13-cis-to-all-trans thermal isomerization within 10−5 s.6 Hence, the photoreaction of the protonated state of TAT rhodopsin is significantly different from other microbial rhodopsins. Interestingly, the deprotonated UV-absorbing state of TAT rhodopsin shows a photocycle lasting ∼100 s with several photointermediates.7 The long photocycle suggests that the UV-absorbing deprotonated state is the functional state driving pH-dependent signal transduction in HIMB114 cells.

Despite detailed biophysical molecular characterization and mechanistic studies, TAT rhodopsin from HIMB114 (TATHIMB) has thus far remained an isolated case of an unusually behaving protein. Here, we identified new rhodopsins homologous to TATHIMB in metagenomic databases which enabled us to study the molecular universality and individuality of characteristics revealed so far in TATHIMB. Seven proteins from this group with diverse TM3 motifs (i.e., TAT, TAI, and TST) could be successfully expressed in E. coli and showed absorption spectra with two peaks at visible and UV wavelength regions similar to TATHIMB. Based on these absorption spectra characteristics in this group, we propose to classify them as “TWin-peaked Rhodopsin (TwR)” family. We compared the amino acid sequences of TwRs and found that a glutamic acid in TM2 (Glu54) is conserved in all of them. Substitution of Glu54 with a glutamine resulted in an increase in the pKa of the RSB, suggesting that the side chain of Glu54 is located near the Schiff base and regulates the RSB pKa value. In addition, the E54Q mutation also affected the oligomeric structures and photoreactions of TwRs. These results demonstrate multiple roles of Glu54 in unique characteristics of TwRs.