Strongly hydrogen-bonded water molecule present near the retinal chromophore of Leptosphaeria rhodopsin, the bacteriorhodopsin-like proton pump from a eukaryote

Leptosphaeria rhodopsin (LR) is an archaeal-type rhodopsin found in fungi, and is the first light-driven proton-pumping retinal protein from eukaryotes. LR pumps protons in a manner similar to that of bacteriorhodopsin (BR), a light-driven proton pump of haloarchaea. The amino acid sequence of LR is more homologous to that of Neurospora rhodopsin (NR) than BR, whereas NR has no proton-pumping activity. These facts raise the question of how the proton-pumping function is achieved. In this paper, we studied structural changes of LR following the retinal photoisomerization by means of low-temperature Fourier transform infrared (FTIR) spectroscopy, and compared the obtained spectra with those for BR and NR. While the light-induced photoisomerization from the all-trans to 13-cis form was commonly observed among LR, BR, and NR, we found that the structural changes of LR are closer to those of BR than to those of NR in terms of detailed vibrational bands of retinal and protein. The most prominent difference was seen for the water O-D stretching vibrations (measured in D2O). LR exhibits an O-D stretch of water at 2257 cm(-1), indicating the presence of a strongly hydrogen-bonded water molecule. Such strongly hydrogen-bonded water molecules (O-D stretch at <2400 cm(-1)) were observed for BR, but not for NR. Comprehensive studies of BR mutants and archaeal rhodopsins have revealed that strongly hydrogen-bonded water molecules are found only in the proteins exhibiting proton-pumping activity, suggesting that strongly hydrogen-bonded water molecules and transient weakening of their binding are essential for the proton-pumping function of rhodopsins. This observation for LR provided additional experimental evidence of the correlation between strongly hydrogen-bonded water molecules and proton-pumping activity of archaeal rhodopsins.     

Structural characterization of proton-pumping rhodopsin lacking a cytoplasmic proton donor residue by X-ray crystallography

DTG/DTS rhodopsin, which was named based on a three-residue motif (DTG or DTS) that is important for its function, is a light-driven proton-pumping microbial rhodopsin using a retinal chromophore. In contrast to other light-driven ion-pumping rhodopsins, DTG/DTS rhodopsin does not have a cytoplasmic proton donor residue, such as Asp, Glu, or Lys. Because of the lack of cytoplasmic proton donor residue, proton directly binds to the retinal chromophore from the cytoplasmic solvent. However, mutational experiments that showed the complicated effects of mutations were not able to clarify the roles played by each residue, and the detail of proton uptake pathway is unclear because of the lack of structural information. To understand the proton transport mechanism of DTG/DTS rhodopsin, here we report the three-dimensional structure of one of the DTG/DTS rhodopsins, PspR from Pseudomonas putida, by X-ray crystallography. We show that the structure of the cytoplasmic side of the protein is significantly different from that of bacteriorhodopsin, the best-characterized proton-pumping rhodopsin, and large cytoplasmic cavities were observed. We propose that these hydrophilic cytoplasmic cavities enable direct proton uptake from the cytoplasmic solvent without the need for a specialized cytoplasmic donor residue. The introduction of carboxylic residues homologous to the cytoplasmic donors in other proton-pumping rhodopsins resulted in higher pumping activity with less pH dependence, suggesting that DTG/DTS rhodopsins are advantageous for producing energy and avoiding intracellular alkalization in soil and plant-associated bacteria.     

Halobacterial rhodopsins

Following the discovery of the bacteriorhodopsin proton pump in Halobacterium halobium (salinarum), not only the halorhodopsin halide pump and two photosensor rhodopsins (sensory rhodopsin and phoborhodopsin) in the same species, but also homologs of these four rhodopsins in strains of other genera of Halobacteriaceae have been reported. Twenty-eight full (and partial) sequences of the genomic DNA of these rhodopsins have been analyzed. The deduced amino acid sequences have led to new strategies and tactics for understanding bacterial rhodopsins on a comparative basis, as summarized briefly in this article. The data discussed include (i) alignment of the sequences to qualify/characterize the conserved residues; (ii) assignment of residues that cause differences in function(s)/properties; and (iii) phylogeny of the halobacterial rhodopsins to suggest their evolutionary paths. The four kinds of rhodopsin in each strain are assumed, on the basis of their genera-specific distributions, to have arisen by at least two gene-duplication processes during evolution prior to generic speciation. The first duplication of the rhodopsin ancestor gene yielded two genes, each of which was duplicated again to give four genes in the ancestor halobacterium. The bacterium carrying four rhodopsin genes, after accumulating mutations, became ready for generic speciation and the delivery of four rhodopsins to each species. The original rhodopsin ancestor is speculated to be closest to the proton pump (bacteriorhodopsin).     

Eubacterial rhodopsins — Unique photosensors and diverse ion pumps

Since the discovery of proteorhodopsins, the ubiquitous marine light-driven proton pumps of eubacteria, a large number of other eubacterial rhodopsins with diverse structures and functions have been characterized. Here, we review the body of knowledge accumulated on the four major groups of eubacterial rhodopsins, with the focus on their biophysical characterization. We discuss advances and controversies on the unique eubacterial sensory rhodopsins (as represented by Anabaena sensory rhodopsin), proton-pumping proteorhodopsins and xanthorhodopsins, as well as novel non-proton ion pumps. This article is part of a Special Issue entitled: Retinal Proteins — You can teach an old dog new tricks.     

Conformational coupling between the cytoplasmic carboxylic acid and the retinal in a fungal light-driven proton pump

Many fungal rhodopsins, eukaryotic structural homologues of the archaeal light-driven proton pump bacteriorhodopsin, have been discovered in the course of genome sequencing projects. Recently, two fungal rhodopsins were characterized in vitro and exhibited very different photochemical behavior. Neurospora rhodopsin possesses a slow photocycle and shows no ion transport, reminiscent of sensory rhodopsins, while Leptosphaeria rhodopsin has a fast bacteriorhodopsin-like photocycle and pumps protons light-dependently. Such a dramatic difference is surprising considering the very high degree of sequence homology of the two proteins. In this paper, we investigate whether the chemical structure of a cytoplasmic carboxylic acid, the homologue of Asp-96 of bacteriorhodopsin serving as a proton donor for the retinal Schiff base, can define the photochemical properties of fungal rhodopsins. We studied mutants of Leptosphaeria rhodopsin in which this aspartic acid was replaced with Glu or Asn using spectroscopy in the infrared and visible ranges. We show that Glu at this position is inefficient as a proton donor similar to a nonprotonatable Asn. Moreover, this replacement induces long-range structural perturbations of the retinal environment, as evidenced by changes in the vibrational bands of retinal (especially, hydrogen-out-of-plane modes) and neighboring aspartic acids and water molecules. The conformational coupling of the mutation site to the retinal may be mediated by helical rearrangements as suggested by the changes in amide and proline vibrational bands. We conclude that the difference in the photochemical behavior of fungal rhodopsins from Leptosphaeria and Neurospora may be ascribed, to some extent, to the replacement of the cytoplasmic proton donor Asp with Glu.     

A microbial rhodopsin with a unique retinal composition shows both sensory rhodopsin II and bacteriorhodopsin-like properties

Rhodopsins possess retinal chromophore surrounded by seven transmembrane α-helices, are widespread in prokaryotes and in eukaryotes, and can be utilized as optogenetic tools. Although rhodopsins work as distinctly different photoreceptors in various organisms, they can be roughly divided according to their two basic functions, light-energy conversion and light-signal transduction. In microbes, light-driven proton transporters functioning as light-energy converters have been modified by evolution to produce sensory receptors that relay signals to transducer proteins to control motility. In this study, we cloned and characterized two newly identified microbial rhodopsins from Haloquadratum walsbyi. One of them has photochemical properties and a proton pumping activity similar to the well known proton pump bacteriorhodopsin (BR). The other, named middle rhodopsin (MR), is evolutionarily transitional between BR and the phototactic sensory rhodopsin II (SRII), having an SRII-like absorption maximum, a BR-like photocycle, and a unique retinal composition. The wild-type MR does not have a light-induced proton pumping activity. On the other hand, a mutant MR with two key hydrogen-bonding residues located at the interaction surface with the transducer protein HtrII shows robust phototaxis responses similar to SRII, indicating that MR is potentially capable of the signaling. These results demonstrate that color tuning and insertion of the critical threonine residue occurred early in the evolution of sensory rhodopsins. MR may be a missing link in the evolution from type 1 rhodopsins (microorganisms) to type 2 rhodopsins (animals), because it is the first microbial rhodopsin known to have 11-cis-retinal similar to type 2 rhodopsins.     

Structural basis of diversification of fungal retinal proteins probed by site-directed mutagenesis of Leptosphaeria rhodopsin

Numerous fungal genomes encode homologs of bacteriorhodopsin (BR), but only two fungal rhodopsins were overexpressed and characterized spectroscopically. Neurospora rhodopsin (NR) is a slow-cycling sensory rhodopsin-like protein, while Leptosphaeria rhodopsin (LR) is a BR-like proton pump. Recently, we found that a conservative replacement of the cytoplasmic proton donor Asp150 by Glu converts LR into an NR-like protein. In this work, we search for structural reasons for the dramatic differences in their photochemistry by mutating the hydrogen-bonding partner of Asp150 (Thr87) and three additional residues (Thr233, Asp248, and Gly271) selected by comparison of the primary structures of NR and LR. We conclude that while these residues may contribute to the differences between LR and NR, they are not crucial for the optimization of the Schiff base reprotonation by Asp150, and that the dramatic effect of the D150E mutation is not a simple result of the introduction of a bulkier glutamate sidechain.     

Rhodopsin-mediated photoreception in cryptophyte flagellates

We show that phototaxis in cryptophytes is likely mediated by a two-rhodopsin-based photosensory mechanism similar to that recently demonstrated in the green alga Chlamydomonas reinhardtii, and for the first time, to our knowledge, report spectroscopic and charge movement properties of cryptophyte algal rhodopsins. The marine cryptophyte Guillardia theta exhibits positive phototaxis with maximum sensitivity at 450 nm and a secondary band above 500 nm. Variability of the relative sensitivities at these wavelengths and light-dependent inhibition of phototaxis in both bands by hydroxylamine suggest the involvement of two rhodopsin photoreceptors. In the related freshwater cryptophyte Cryptomonas sp. two photoreceptor currents similar to those mediated by the two sensory rhodopsins in green algae were recorded. Two cDNA sequences from G. theta and one from Cryptomonas encoding proteins homologous to type 1 opsins were identified. The photochemical reaction cycle of one Escherichia-coli-expressed rhodopsin from G. theta (GtR1) involves K-, M-, and O-like intermediates with relatively slow (approximately 80 ms) turnover time. GtR1 shows lack of light-driven proton pumping activity in E. coli cells, although carboxylated residues are at the positions of the Schiff base proton acceptor and donor as in proton pumping rhodopsins. The absorption spectrum, corresponding to the long-wavelength band of phototaxis sensitivity, makes this pigment a candidate for one of the G. theta sensory rhodopsins. A second rhodopsin from G. theta (GtR2) and the one from Cryptomonas have noncarboxylated residues at the donor position as in known sensory rhodopsins.     

Diversity of bacteriorhodopsins in different hypersaline waters from a single Spanish saltern

Haloarchaeal rhodopsins are a diverse group of transmembrane proteins that use light energy to drive several different cellular processes. Two rhodopsins, bacteriorhodopsin and halorhodopsins, are H+ and Cl- ion pumps, respectively, and two rhodopsins, sensory rhodopsin I and II, regulate phototaxis. Bacteriorhodopsin is of special interest as it is a non-chlorophyll-based type of phototrophy (i.e. generation of chemical energy from light energy). However, very little is known about the diversity and distribution of rhodopsin genes in hypersaline environments. Here, we have used environmental PCR and cloning techniques to directly retrieve rhodopsin genes from three different salinity ponds located in a sea salt manufacturing facility near Alicante, Spain. Our survey resulted in the discovery of previously concealed variation including what is hypothesized to be bacteriorhodopsin genes from the uncultivated square morphotype that dominates these environments. In some instances, identical genes were discovered in seemingly different habitats suggesting that some haloarchaea are present over widely varying concentrations of salt.     

Hydrogen-bonding changes of internal water molecules upon the actions of microbial rhodopsins studied by FTIR spectroscopy

Microbial rhodopsins are classified into type-I rhodopsins, which utilize light energy to perform wide varieties of function, such as proton pumping, ion pumping, light sensing, cation channels, and so on. The crystal structures of several type-I rhodopsins were solved and the molecular mechanisms have been investigated based on the atomic structures. However, the crystal structures of proteins of interest are not always available and the basic architectures are sometimes quite similar, which obscures how the proteins achieve different functions. Stimulus-induced difference FTIR spectroscopy is a powerful tool to detect minute structural changes providing a clue for elucidating the molecular mechanisms. In this review, the studies on type-I rhodopsins from fungi and marine bacteria, whose crystal structures have not been solved yet, were summarized. Neurospora rhodopsin and Leptosphaeria rhodopsin found from Fungi have sequence similarity. The former has no proton pumping function, while the latter has. Proteorhodopsin is another example, whose proton pumping machinery is altered at alkaline and acidic conditions. We described how the structural changes of protein were different and how water molecules were involved in them. We reviewed the results on dynamics of the internal water molecules in pharaonis halorhodopsin as well. This article is part of a Special Issue entitled: Retinal Proteins — You can teach an old dog new tricks.     

Conformational changes in the photocycle of Anabaena sensory rhodopsin: absence of the Schiff base counterion protonation signal

Anabaena sensory rhodopsin (ASR) is a novel microbial rhodopsin recently discovered in the freshwater cyanobacterium Anabaena sp. PCC7120. This protein most likely functions as a photosensory receptor as do the related haloarchaeal sensory rhodopsins. However, unlike the archaeal pigments, which are tightly bound to their cognate membrane-embedded transducers, ASR interacts with a soluble cytoplasmic protein analogous to transducers of animal vertebrate rhodopsins. In this study, infrared spectroscopy was used to examine the molecular mechanism of photoactivation in ASR. Light adaptation of the pigment leads to a phototransformation of an all-trans/15-anti to 13-cis/15-syn retinylidene-containing species very similar in chromophore structural changes to those caused by dark adaptation in bacteriorhodopsin. Following 532 nm laser-pulsed excitation, the protein exhibits predominantly an all-trans retinylidene photocycle containing a deprotonated Schiff base species similar to those of other microbial rhodopsins such as bacteriorhodopsin, sensory rhodopsin II, and Neurospora rhodopsin. However, no changes are observed in the Schiff base counterion Asp-75, which remains unprotonated throughout the photocycle. This result along with other evidence indicates that the Schiff base proton release mechanism differs significantly from that of other known microbial rhodopsins, possibly because of the absence of a second carboxylate group at the ASR photoactive site. Several conformational changes are detected during the ASR photocycle including in the transmembrane helices E and G as indicated by hydrogen-bonding alterations of their native cysteine residues. In addition, similarly to animal vertebrate rhodopsin, perturbations of the polar head groups of lipid molecules are detected.     

Bacteriorhodopsin-like proteins of eubacteria and fungi: the extent of conservation of the haloarchaeal proton-pumping mechanism.

A stereotypical image of a retinal-binding proton pump derived from extensive studies of halobacterial ion-transporting and sensory rhodopsins is a fast-cycling protein which possesses two strategically placed carboxylic acids serving as proton donor and acceptor for the retinal Schiff base. We review recent biophysical and bioinformatic data on the novel eubacterial and eucaryotic rhodopsins to analyze the extent of conservation of the haloarchaeal mechanism of transmembrane proton transport. We show that only the most essential elements of the haloarchaeal proton-pumping machinery are conserved universally, and that a mere presence of these elements in primary structures does not guarantee the proton-pumping ability.     

Actinorhodopsins: proteorhodopsin-like gene sequences found predominantly in non-marine environments

Proteorhodopsins are light-energy-harvesting transmembrane proteins encoded by genes recently discovered in the surface waters of the world's oceans. Metagenomic data from the Global Ocean Sampling expedition (GOS) recovered 2674 proteorhodopsin-related sequences from 51 aquatic samples. Four of these samples were from non-marine environments, specifically, Lake Gatun within the Panama Canal, Delaware Bay and Chesapeake Bay and the Punta Cormorant Lagoon in Ecuador. Rhodopsins related to but phylogenetically distinct from most sequences designated proteorhodopsins were present at all four of these non-marine sites and comprised three different clades that were almost completely absent from marine samples. Phylogenomic analyses of genes adjacent to those encoding these novel rhodopsins suggest affiliation to the Actinobacteria , and hence we propose to name these divergent, non-marine rhodopsins 'actinorhodopsins'. Actinorhodopsins conserve the acidic amino acid residues critical for proton pumping and their genes lack genomic association with those encoding photo-sensory transducer proteins, thus supporting a putative ion pumping function. The ratio of rec A and rad A to rhodopsin genes in the different environment types sampled within the GOS indicates that rhodopsins of one type or another are abundant in microbial communities in freshwater, estuarine and lagoon ecosystems, supporting an important role for these photosystems in all aquatic environments influenced by sunlight.     

Substitution of Pro206 and Ser86 residues in the retinal binding pocket of Anabaena sensory rhodopsin is not sufficient for proton pumping function

Anabaena sensory rhodopsin is a seven transmembrane protein that uses all-trans/13-cis retinal as a chromophore. About 22 residues in the retinal-binding pocket of microbial rhodopsins are conserved and important to control the quality of absorbing light and the function of ion transport or sensory transduction. The absorption maximum is 550 nm in the presence of all-trans retinal at dark. Here, we mutated Pro206 to Glu or Asp, of which the residue is conserved as Asp among all other microbial rhodopsins, and the absorption maximum and pKa of the proton acceptor group were measured by absorption spectroscopy at various pHs. Anabaena rhodopsin was expressed best in Escherichia coli in the absence of extra leader sequence when exogenous all-trans retinal was added. The wild-type Anabaena rhodopsin showed small absorption maximum changes between pH 4 and 11. In addition, Pro206Asp showed 46 nm blue-shift at pH 7.0. Pro206Glu or Asp may change the contribution to the electron distribution of the retinal that is involved in the major role of color tuning for this pigment. The critical residue Ser86 (Asp 96 position in bacteriorhodopsin: proton donor) for the pumping activity was replaced with Asp, but it did not change the proton pumping activity of Anabaena rhodopsin.     

New Insights into the Evolutionary History of Type 1 Rhodopsins

Type 1 (archaeal) rhodopsins and related rhodopsin-like proteins had been described in a few halophile archaea, -proteobacteria, a single cyanobacteria, some fungi, and a green alga. In exhaustive database searches, we detected rhodopsin-related sequences derived not only from additional fungal species but also from organisms belonging to three groups in which opsins had hitherto not been described: the -proteobacterium Magnetospirillum magnetotacticum, the cryptomonad alga Guillardia theta, and the dinoflagellate Pyrocystis lunula. Putative plant and human type 1 rhodopsin sequences found in the databases are demonstrated to be contaminants of fungal origin. However, a highly diverged sequence supposedly from the plant Oryza sativa was found that is, together with the Pyrocystis sequence, quite similar to -proteobacterial rhodopsins. These close relationships suggest that at least one horizontal gene transfer event involving rhodopsin genes occurred between prokaryotes and eukaryotes. Alternative hypotheses to explain the current phylogenetic range of type 1 rhodopsins are suggested. The broader phylogenetic range found is compatible with an ancient origin of type 1 rhodopsins, their patchy distribution being caused by losses in multiple lineages. However, the possibility of ancient horizontal transfer events between distant relatives cannot be dismissed.     

Characterization of the chimeric seven-transmembrane protein containing conserved region of helix C–F of microbial rhodopsin from Ganges River

Proteorhodopsin (PR) is a light-driven proton pump that has been found in a variety of marine bacteria. Recently, many PR-like genes were found in non-marine environments. The goal of this study is to explore the function of rhodopsins that exist only as partial proteo-opsin genes using chimeras with marine green PR (GPR). We isolated nine partial genes of PR homologues using polymerase chain reaction (PCR) and chose three homologues of GPR from the surface of the Ganges River, which has earned them the name “CFR, Chimeric Freshwater Rhodopsin.” In order to characterize the proteins, we constructed the cassette based on GPR sequence without helices C to F and inserted the isolated conserved partial sequences. When expressed in E. coli, we could observe light-driven proton pumping activity similar to proteorhodopsin, however, photocycle kinetics of CFRs are much slower than proteorhodopsin. Half-time decay of O intermediates of CFRs ranged between 143 and 333 ms at pH 10; their absorption maxima were between 515 and 522 nm at pH 7. We can guess that the function of native rhodopsin, a retinal protein of fresh water bacteria, may be a light-driven proton transport based on the results from chimeric freshwater rhodopsins. This approach will enable many labs that keep reporting partial PCR-based opsin sequences to finally characterize their proteins.     

Light-induced intramolecular charge movements in microbial rhodopsins in intact E. coli cells.

Microbial rhodopsins undergo cyclic photochemical reactions (photocycles) in which proton transfers and conformational changes result in charge displacements during transitions between photocycle intermediates. We report a new photoelectric method to monitor charge movements during rhodopsin photocycling with fast kinetic resolution in suspensions of intact E. coli cells. The method monitors electrical currents resulting from asymmetric photoexcitation of microbial rhodopsins by a unilateral laser flash, and kinetically resolves intramolecular charge movements. We investigated E. coli-expressed proton-transporting rhodopsins, specifically green- and blue-absorbing proteorhodopsins (GPR and BPR, respectively) from uncultivated marine plankton, and sensory rhodopsins, namely receptors from Natronomonas pharaonis and Anabaena (Nostoc) sp. PCC7120. Kinetic components of the currents correlate with photochemical transformations of the pigments, and the integrated current measures net transport by the proton-pumping rhodopsins. The photoelectric measurements distinguish between known light-driven transporters and photosensors, and reveal differences in proton transfer reactions in the two tested proton pumps. Screening of nine newly identified proteorhodopsins reveals two with GPR-type charge movements, five with BPR-type, and two with the characteristics of the sensory rhodopsins. The approach developed in the present work provides a direct, rapid and informative method for studying electrogenic events in rhodopsin photocycles and also gives a clue to functions of newly found microbial rhodopsins in nature.     

Photophosphorylation elements in halobacteria: An A-type ATP synthase and bacterial rhodopsins

Photophosphorylation in halobacteria is carried out by two rather simple elements: an A-type ATP synthase and light-driven ion-pumping bacterial rhodopsins. The unique features of halobacterial ATP synthase, mostly common to archaebacteria (A-type), and of new members of the bacteriorhodopsin family are introduced along with studies performed in the authors' laboratory. This is the story of how we found that the A-type ATP synthase is close to V-type ATPase but far from F-type ATPase, although all three ATPases are believed to have the same ancestor. Archaerhodopsins, the new members of the proton-pumping retinal proteins, were found in Australian halobacteria and have been used in a comparative study of bacterial rhodopsins.     

Isolation and structure of a rhodopsin gene from D. melanogaster

Using a novel method for detecting cross-homologous nucleic acid sequences we have isolated the gene coding for the major rhodopsin of Drosophila melanogaster and mapped it to chromosomal region 92B8-11. Comparison of cDNA and genomic DNA sequences indicates that the gene is divided into five exons. The amino acid sequence deduced from the nucleotide sequence is 373 residues long, and the polypeptide chain contains seven hydrophobic segments that appear to correspond to the seven transmembrane segments characteristic of other rhodopsins. Three regions of Drosophila rhodopsin are highly conserved with the corresponding domains of bovine rhodopsin, suggesting an important role for these polypeptide regions.