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.

Thermophilic rhodopsin (TR) is a light-driven proton pump from the extreme thermophile Thermus thermophilus JL-18. Previous studies on TR solubilized with detergent showed that the protein exhibits high thermal stability and forms a trimer at room temperature but irreversibly dissociates into monomers when incubated at physiological temperature (75 °C). In the present study, we used resonance Raman (RR) spectroscopy, solid-state NMR spectroscopy, and high-speed atomic force microscopy to analyze the oligomeric structure of TR in a lipid environment. The obtained spectra and microscopic images demonstrate that TR adopts a pentameric form in a lipid environment and that this assembly is stable at the physiological temperature, in contrast to the behavior of the protein in the solubilized state. These results indicate that the thermal stability of the oligomeric assembly of TR is higher in a lipid environment than in detergent micelles. The observed RR spectra also showed that the retinal chromophore is strongly hydrogen bonded to an internal water molecule via a protonated Schiff base, which is characteristic of proton-pumping rhodopsins. The obtained data strongly suggest that TR functions in the pentameric form at physiological temperature in the extreme thermophile T. thermophilus JL-18. We utilized the high thermal stability of the monomeric form of solubilized TR and here report the first RR spectra of the monomeric form of a microbial rhodopsin. The observed RR spectra revealed that the monomerization of TR alters the chromophore structure: there are changes in the bond alternation of the polyene chain and in the hydrogen-bond strength of the protonated Schiff base. The present study revealed the high thermal stability of oligomeric assemblies of TR in the lipid environment and suggested the importance of using TR embedded in lipid membrane for elucidation of its functional mechanism.

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.

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