Many organisms capture or sense sunlight using rhodopsin pigments, which are integral membrane proteins that bind retinal chromophores. Rhodopsins comprise two distinct protein families , type-1 (microbial rhodopsins) and type-2 (animal rhodopsins). The two families share similar topologies and contain seven transmembrane helices that form a pocket in which retinal is linked covalently as a protonated Schiff base to a lysine at the seventh transmembrane helix. Type-1 and type-2 rhodopsins show little or no sequence similarity to each other, as a consequence of extensive divergence from a common ancestor or convergent evolution of similar structures . Here we report a previously unknown and diverse family of rhodopsins-which we term the heliorhodopsins-that we identified using functional metagenomics and that are distantly related to type-1 rhodopsins. Heliorhodopsins are embedded in the membrane with their N termini facing the cell cytoplasm, an orientation that is opposite to that of type-1 or type-2 rhodopsins. Heliorhodopsins show photocycles that are longer than one second, which is suggestive of light-sensory activity. Heliorhodopsin photocycles accompany retinal isomerization and proton transfer, as in type-1 and type-2 rhodopsins, but protons are never released from the protein, even transiently. Heliorhodopsins are abundant and distributed globally; we detected them in Archaea, Bacteria, Eukarya and their viruses. Our findings reveal a previously unknown family of light-sensing rhodopsins that are widespread in the microbial world.

Light-driven outward H+ pumps are widely distributed in nature, converting sunlight energy into proton motive force. Here we report the characterization of an oppositely directed H+ pump with a similar architecture to outward pumps. A deep-ocean marine bacterium, Parvularcula oceani, contains three rhodopsins, one of which functions as a light-driven inward H+ pump when expressed in Escherichia coli and mouse neural cells. Detailed mechanistic analyses of the purified proteins reveal that small differences in the interactions established at the active centre determine the direction of primary H+ transfer. Outward H+ pumps establish strong electrostatic interactions between the primary H+ donor and the extracellular acceptor. In the inward H+ pump these electrostatic interactions are weaker, inducing a more relaxed chromophore structure that leads to the long-distance transfer of H+ to the cytoplasmic side. These results demonstrate an elaborate molecular design to control the direction of H+ transfers in proteins.

The naturally occurring channelrhodopsin variant anion channelrhodopsin-1 (ACR1), discovered in the cryptophyte algae Guillardia theta, exhibits large light-gated anion conductance and high anion selectivity when expressed in heterologous settings, properties that support its use as an optogenetic tool to inhibit neuronal firing with light. However, molecular insight into ACR1 is lacking owing to the absence of structural information underlying light-gated anion conductance. Here we present the crystal structure of G. theta ACR1 at 2.9 Å resolution. The structure reveals unusual architectural features that span the extracellular domain, retinal-binding pocket, Schiff-base region, and anion-conduction pathway. Together with electrophysiological and spectroscopic analyses, these findings reveal the fundamental molecular basis of naturally occurring light-gated anion conductance, and provide a framework for designing the next generation of optogenetic tools.

Channelrhodopsin (ChR) functions as a light-gated ion channel in Chlamydomonas reinhardtii. Passive transport of cations by ChR is fundamentally different from the active transport by light-driven ion pumps such as archaerhodopsin, bacteriorhodopsin, and halorhodopsin. These microbial rhodopsins are important tools for optogenetics, where ChR is used to activate neurons by light, while the ion pumps are used for neural silencing. Ion-transport functions by these rhodopsins strongly depend on the specific hydrogen-bonding networks containing water near the retinal chromophore. In this work, we measured protein-bound water molecules in a chimeric ChR protein of ChR1 (helices A to E) and ChR2 (helices F and G) of Chlamydomonas reinhardtii using low-temperature FTIR spectroscopy at 77 K. We found that the active center of ChR possesses more water molecules (9 water vibrations) than those of other microbial (2-6 water vibrations) and animal (6-8 water vibrations) rhodopsins. We conclude that the protonated retinal Schiff base interacts with the counterion (Glu162) directly, without the intervening water molecule found in proton-pumping microbial rhodopsins. The present FTIR results and the recent X-ray structure of ChR reveal a unique hydrogen-bonding network around the active center of this light-gated ion channel.

The recently identified Krokinobacter rhodopsin 2 (KR2) functions as a light-driven sodium ion pump. The structure of the retinal-binding pocket of KR2 offers important insights into the mechanisms of KR2, which has motif of Asn112, Asp116, and Gln123 (NDQ) that is common among sodium ion pump rhodopsins but is unique among other microbial rhodopsins. Here we present solid-state nuclear magnetic resonance (NMR) characterization of retinal and functionally important residues in the vicinity of retinal in the ground state. We assigned chemical shifts of retinal C14 and C20 atoms, and Tyr218Cζ, Lys255Cε, and the protonated Schiff base of KR2 in lipid environments at acidic and neutral pH. N NMR signals of the protonated Schiff base showed a twist around the N-Cε bond under neutral conditions, compared with other microbial rhodopsins. These data indicated that the location of the counterion Asp116 is one helical pitch toward the cytoplasmic side. In acidic environments, theN Schiff base signal was shifted to a lower field, indicating that protonation of Asp116 induces reorientation during interactions between the Schiff base and Asp116. In addition, the Tyr218 residue in the vicinity of retinal formed a weak hydrogen bond with Asp251, a temporary Na-binding site during the photocycle. These features may indicate unique mechanisms of sodium ion pumps.

Three kinds of photochemical reactions are known in flavins as chromophores of photosensor proteins, reflecting the various catalytic reactions of the flavin in flavoenzymes. Sensor of blue light using the flavin FAD (BLUF) domains exhibit a unique photoreaction compared with other flavin-binding photoreceptors in that the chromophore does not change its chemical structure between unphotolyzed and intermediate states. Rather, the hydrogen bonding environment is altered, whereby the conserved Gln and Tyr residues near FAD play a crucial role. One proposal for this behavior is that the conserved Gln changes its chemical structure from a keto to an enol. We applied light-induced difference Fourier transform infrared (FTIR) spectroscopy to AppA-BLUF. The spectra of AppA-BLUF exhibited a different feature upon N-Gln labeling compared with the previously reported spectra from BlrB, a different BLUF domain. The FTIR signals were interpreted from quantum mechanics/molecular mechanics (QM/MM) calculation as the keto-enol tautomerization and rotation of the Gln63 side chain in the AppA-BLUF domain. The former was consistent with the result from BlrB, but the latter was not uniquely determined by the previous study. QM/MM calculation also indicated that the infrared signal shape is influenced depending on whether a Trp side chain forms a hydrogen bond with the Gln side chain. FTIR spectra and QM/MM simulations concluded that Trp104 does not flip out but is maintained in the intermediate state. In contrast, our data revealed that the Trp residue at the corresponding position in BlrB faces outward in both states.

Both designed and natural anion-conducting channelrhodopsins (dACRs and nACRs, respectively) have been widely applied in optogenetics (enabling selective inhibition of target-cell activity during animal behaviour studies), but each class exhibits performance limitations, underscoring trade-offs in channel structure-function relationships. Therefore, molecular and structural insights into dACRs and nACRs will be critical not only for understanding the fundamental mechanisms of these light-gated anion channels, but also to create next-generation optogenetic tools. Here we report crystal structures of the dACR iC++, along with spectroscopic, electrophysiological and computational analyses that provide unexpected insights into pH dependence, substrate recognition, channel gating and ion selectivity of both dACRs and nACRs. These results enabled us to create an anion-conducting channelrhodopsin integrating the key features of large photocurrent and fast kinetics alongside exclusive anion selectivity.

One of the main functions of microbial rhodopsins is outward-directed light-driven proton transport across the plasma membrane, which can provide sources of energy alternative to respiration and chlorophyll photosynthesis. Proton-pumping rhodopsins are found in Archaea (Halobacteria), multiple groups of Bacteria, numerous fungi, and some microscopic algae. An overwhelming majority of these proton pumps share the common transport mechanism, in which a proton from the retinal Schiff base is first transferred to the primary proton acceptor (normally an Asp) on the extracellular side of retinal. Next, reprotonation of the Schiff base from the cytoplasmic side is mediated by a carboxylic proton donor (Asp or Glu), which is located on helix C and is usually hydrogen-bonded to Thr or Ser on helix B. The only notable exception from this trend was recently found in Exiguobacterium, where the carboxylic proton donor is replaced by Lys. Here we describe a new group of efficient proteobacterial retinal-binding light-driven proton pumps which lack the carboxylic proton donor on helix C (most often replaced by Gly) but possess a unique His residue on helix B. We characterize the group spectroscopically and propose that this histidine forms a proton-donating complex compensating for the loss of the carboxylic proton donor.