The preferential transport of NO3- by full-length Guillardia theta anion channelrhodopsin 1 is enhanced by its extended cytoplasmic domain.

Previous research of anion channelrhodopsins (ACRs) has been performed using cytoplasmic domain (CPD)-deleted constructs and therefore have overlooked the native functions of full-length ACRs and the potential functional role(s) of the CPD. In this study, we used the recombinant expression of full-length Guillardia theta ACR1 (GtACR1_full) for pH measurements in Pichia pastoris cell suspensions as an indirect method to assess its anion transport activity and for absorption spectroscopy and flash photolysis characterization of the purified protein. The results show that the CPD, which was predicted to be intrinsically disordered and possibly phosphorylated, enhanced NO3- transport compared to Cl- transport, which resulted in the preferential transport of NO3-. This correlated with the extended lifetime and large accumulation of the photocycle intermediate that is involved in the gate-open state. Considering that the depletion of a nitrogen source enhances the expression of GtACR1 in native algal cells, we suggest that NO3- transport could be the natural function of GtACR1_full in algal cells.

Multistep conformational changes leading to the gate opening of light-driven sodium pump rhodopsin.

Membrane transport proteins require a gating mechanism that opens and closes the substrate transport pathway to carry out unidirectional transport. The "gating" involves large conformational changes and is achieved via multistep reactions. However, these elementary steps have not been clarified for most transporters due to the difficulty of detecting the individual steps. Here, we propose these steps for the gate opening of the bacterial Na+ pump rhodopsin, which outwardly pumps Na+ upon illumination. We herein solved an asymmetric dimer structure of Na+ pump rhodopsin from the bacterium Indibacter alkaliphilus. In one protomer, the Arg108 sidechain is oriented toward the protein center and appears to block a Na+ release pathway to the extracellular (EC) medium. In the other protomer, however, this sidechain swings to the EC side and then opens the release pathway. Assuming that the latter protomer mimics the Na+-releasing intermediate, we examined the mechanism for the swing motion of the Arg108 sidechain. On the EC surface of the first protomer, there is a characteristic cluster consisting of Glu10, Glu159, and Arg242 residues connecting three helices. In contrast, this cluster is disrupted in the second protomer. Our experimental results suggested that this disruption is a key process. The cluster disruption induces the outward movement of the Glu159-Arg242 pair and simultaneously rotates the seventh transmembrane helix. This rotation resultantly opens a space for the swing motion of the Arg108 sidechain. Thus, cluster disruption might occur during the photoreaction and then trigger sequential conformation changes leading to the gate-open state.

Elucidation of a Thermodynamical Feature Attributed to Substrate Binding to the Prokaryotic H+/Oligopeptide Cotransporter YdgR with Calorimetric Analysis: The Substrate Binding Driven by the Change in Entropy Implies the Release of Bound Water Molecules from the Binding Pocket.

Here, we have elucidated the substrate recognition mechanism by a prokaryotic H+/oligopeptide cotransporter, YdgR, using isothermal titration calorimetry. Under acidic conditions (pH 6.0), the binding of a dipeptide, Val-Ala, to YdgR elicited endothermic enthalpy, which compensated for the increase in entropy due to dipeptide binding. A series of dipeptides were used in the binding titration. The dipeptides represent Val-X and X-Val, where X is Ala, Ser, Val, Tyr, or Phe. Most dipeptides revealed endothermic enthalpy, which was completely compensated by the increase in entropy due to dipeptide binding. The change in enthalpy due to binding correlated well with the change in entropy, whereas the Gibbs free energy involved in the binding of the dipeptide to YdgR remained unchanged irrespective of dipeptide sequences, implying that the binding reaction was driven by entropy, that is, the release of bound water molecules in the binding pocket. It is also important to clarify that, based on the prediction of water molecules in the ligand-binding pocket of YdgR, the release of three bound water molecules in the putative substrate binding pocket occurred through binding to YdgR. In the comparison of Val-X and X-Val dipeptides, the N-terminal region of the binding pocket might contain more bound water molecules than the C-terminal region. In light of these findings, we suggest that bound water molecules might play an important role in substrate recognition and binding by YdgR.

Reisomerization of retinal represents a molecular switch mediating Na+ uptake and release by a bacterial sodium-pumping rhodopsin.

Sodium-pumping rhodopsins (NaRs) are membrane transporters that utilize light energy to pump Na+ across the cellular membrane. Within the NaRs, the retinal Schiff base chromophore absorbs light, and a photochemically induced transient state, referred to as the "O intermediate", performs both the uptake and release of Na+. However, the structure of the O intermediate remains unclear. Here, we used time-resolved cryo-Raman spectroscopy under preresonance conditions to study the structure of the retinal chromophore in the O intermediate of an NaR from the bacterium Indibacter alkaliphilus. We observed two O intermediates, termed O1 and O2, having distinct chromophore structures. We show O1 displays a distorted 13-cis chromophore, while O2 contains a distorted all-trans structure. This finding indicated that the uptake and release of Na+ are achieved not by a single O intermediate but by two sequential O intermediates that are toggled via isomerization of the retinal chromophore. These results provide crucial structural insight into the unidirectional Na+ transport mediated by the chromophore-binding pocket of NaRs.

Phenylazothiazoles as Visible-Light Photoswitches.

A novel class of photoswitches based on a phenylazothiazole scaffold that undergoes reversible isomerization under visible-light irradiation is reported. The photoswitch, which comprises a thiazole heteroaryl segment directly connected to a phenyl azo chromophore, has very different spectral characteristics, such as a redshifted absorption maximum wavelength and well-separated absorption bands of the trans and cis isomers, than conventional azobenzene and other heteroaryl azo compounds. Substituents at the ortho and para positions of the phenyl ring of the photoswitch resulted in a further shift to longer wavelengths up to 525 nm at the absorption maximum with a small thermal stability compensation. These photoswitches showed excellent photostationary distributions of the trans and cis isomers, thermal half-lives of up to 7.2 h, and excellent reductant stability. The X-ray crystal structure analysis revealed that the trans isomers exhibited a planar geometry and the cis isomers exhibited a T-shaped orthogonal geometry. Detailed ab initio calculations further demonstrated the plausible electronic transitions and isomerization energy barriers, which were consistent with the experimental observations. The fundamental design principles elucidated in this study will aid in the development of a wide variety of visible-light photoswitches for photopharmacological applications.

Efficient recombinant production of mouse-derived cryptdin family peptides by a novel facilitation strategy for inclusion body formation.

BACKGROUND: A number of antimicrobial peptides (AMPs) hold promise as new drugs owing to their potent bactericidal activity and because they are often refractory to the development of drug resistance. Cryptdins (Crps) are a family of antimicrobial peptides found in the small intestine of mice, comprising six isoforms containing three sets of disulfide bonds. Although Crp4 is actively being investigated, there have been few studies to date on the other Crp isoforms. A prerequisite for detailed characterization of the other Crp isoforms is establishment of efficient sample preparation methods. RESULTS: To avoid degradation during recombinant expression of Crps in E. coli, co-expression of Crps with the aggregation-prone protein human α-lactalbumin (HLA) was used to promote the formation of stable inclusion bodies. Using this method, the production of Crp4 and Crp6 by the BL21 strain was effective, but the expression of other Crp isoforms was not as efficient. The results of a cell-free system study suggested that Crps were degraded, even though a substantial amounts of Crps were synthesized. Therefore, using the Origami™ B strain, we were able to significantly increase the expression efficiency of Crps by promoting the formation of erroneous intermolecular disulfide bonds between HLA and Crps, thereby promoting protein aggregation and inclusion body formation, which prevented degradation. The various Crp isoforms were successfully refolded in vitro and purified using reversed-phase HPLC. In addition, the yield was further improved by deformylation of formyl-Crps. We measured the antibacterial activity of Crps against both Gram-positive and Gram-negative bacteria. Each Crp isoform exhibited a completely different trend in antimicrobial activity, although conformational analysis by circular dichroism did not reveal any significant steric differences. CONCLUSION: In this study, we established a novel and efficient method for the production of the cryptdin family of cysteine-containing antimicrobial peptides. Additionally, we found that there were notable differences in the antibacterial activities of the various Crp family members. The expression system established in this study is expected to provide new insights regarding the mechanisms underlying the different antibacterial activities of the Crp family of peptides.

Real-time identification of two substrate-binding intermediates for the light-driven sodium pump rhodopsin.

Membrane transport proteins undergo critical conformational changes during substrate uptake and release, as the substrate-binding site is believed to switch its accessibility from one side of the membrane to the other. Thus, at least two substrate-binding intermediates should appear during the process, that is, after uptake and before the release of the substrate. However, this view has not been verified for most transporters because of the difficulty in detecting short-lived intermediates. Here, we report real-time identification of these intermediates for the light-driven outward current-generating Na+-pump rhodopsin. We triggered the transport cycle of Na+-pump rhodopsin using a short laser pulse, and subsequent formation and decay of various intermediates was detected by time-resolved measurements of absorption changes. We used this method to analyze transport reactions and elucidated the sequential formation of the Na+-binding intermediates O1 and O2. Both intermediates exhibited red-shifted absorption spectra and generated transient equilibria with short-wavelength intermediates. The equilibria commonly shifted toward O1 and O2 with increasing Na+ concentration, indicating that Na+ is bound to these intermediates. However, these equilibria were formed independently; O1 reached equilibrium with preceding intermediates, indicating Na+ uptake on the cytoplasmic side. In contrast, O2 reached equilibrium with subsequent intermediates, indicating Na+ release on the extracellular side. Thus, there is an irreversible switch in "accessibility" during the O1 to O2 transition, which could represent one of the key processes governing unidirectional Na+ transport.

Replaceability of Schiff base proton donors in light-driven proton pump rhodopsins.

Many H+-pump rhodopsins conserve "H+ donor" residues in cytoplasmic (CP) half channels to quickly transport H+ from the CP medium to Schiff bases at the center of these proteins. For conventional H+ pumps, the donors are conserved as Asp or Glu but are replaced by Lys in the minority, such as Exiguobacterium sibiricum rhodopsin (ESR). In dark states, carboxyl donors are protonated, whereas the Lys donor is deprotonated. As a result, carboxyl donors first donate H+ to the Schiff bases and then capture the other H+ from the medium, whereas the Lys donor first captures H+ from the medium and then donates it to the Schiff base. Thus, carboxyl and Lys-type H+ pumps seem to have different mechanisms, which are probably optimized for their respective H+-transfer reactions. Here, we examined these differences via replacement of donor residues. For Asp-type deltarhodopsin (DR), the embedded Lys residue distorted the protein conformation and did not act as the H+ donor. In contrast, for Glu-type proteorhodopsin (PR) and ESR, the embedded residues functioned well as H+ donors. These differences were further examined by focusing on the activation volumes during the H+-transfer reactions. The results revealed essential differences between archaeal H+ pump (DR) and eubacterial H+ pumps PR and ESR. Archaeal DR requires significant hydration of the CP channel for the H+-transfer reactions; however, eubacterial PR and ESR require the swing-like motion of the donor residue rather than hydration. Given this common mechanism, donor residues might be replaceable between eubacterial PR and ESR.

Potent bactericidal activity of reduced cryptdin-4 derived from its hydrophobicity and mediated by bacterial membrane disruption.

Defensin is a cysteine-rich antimicrobial peptide with three disulphide bonds under normal oxidative conditions. Cryptdin-4 (Crp4) is a defensin secreted by Paneth cells in the small intestine of mice, and only reduced Crp4 (Crp4red) shows activity against enteric commensal bacteria, although both oxidised Crp4 (Crp4ox) and Crp4red can kill non-commensal bacteria. To investigate the molecular factors that affect the potent antimicrobial activity of Crp4red, the bactericidal activities of Crp4ox and Crp4red, Crp4 with all Cys residues substituted with Ser peptide (6C/S-Crp4), and Crp4 with all thiol groups modified by N-ethylmaleimide (NEM-Crp4) were assessed. All peptides showed bactericidal activity against non-commensal bacteria, whereas Crp4red and NEM-Crp4 showed bactericidal activity against commensal bacteria. These potent peptides exhibited high hydrophobicity, which was strongly correlated with membrane insertion. Intriguingly, Crp4ox formed electrostatic interactions with the membrane surface of bacteria, even without exerting bactericidal activity. Moreover, the bactericidal activity of both oxidised and reduced forms of Crp4 was abolished by inhibition of electrostatic interactions; this finding suggests that Crp4red targets bacterial membranes. Finally, a liposome leakage assay against lipids extracted from commensal bacteria demonstrated a correlation with bactericidal activity. These results suggest that the potent bactericidal activity of Crp4red is derived from its hydrophobicity, and the bactericidal mechanism involves disruption of the bacterial membrane. Findings from this study provide a better understanding of the bactericidal mechanism of both Crp4ox and Crp4red.

Low-temperature Raman spectroscopy of sodium-pump rhodopsin from Indibacter alkaliphilus: insight of Na+ binding for active Na+ transport.

We carried out the low-temperature Raman measurement of a sodium pump rhodopsin from Indibacter alkaliphilus (IaNaR) and examined the primary structural change for the light-driven Na+ pump. We observed that photoexcitation of IaNaR produced the distorted 13-cis retinal chromophore in the presence of Na+, while the structural distortion was significantly relaxed in the absence of Na+. This structural difference of the chromophore with/without Na+ was attributed to the Na+ binding to the protein, which alters the active site. Using the spectral sensitivity to the ion binding, we found that IaNaR had a second Na+ binding site in addition to the one already specified on the extracellular surface. To date, the Na+ binding has not been considered as a prerequisite for Na+ transport. However, this study provides insight that the protein structural change induced by the ion binding involved the formation of an R108-D250 salt bridge, which has critical importance in the active transport of Na+.

Protonation State of a Histidine Residue in Human Oligopeptide Transporter 1 (hPEPT1) Regulates hPEPT1-Mediated Efflux Activity.

To clarify the role of an amino acid residue in the pH-dependent efflux process in Chinese hamster ovary (CHO) cells expressing the human oligopeptide transporter hPEPT1 (CHO/hPEPT1), we determined the effect of extracellular pH on the hPEPT1-mediated efflux process. The efflux of glycylsarcosine (Gly-Sar), a typical substrate for hPEPT1, was determined using an infinite dilution method after cells were preloaded with [3H]-Gly-Sar. The efflux of [3H]-Gly-Sar was stimulated by 5 mM unlabeled hPEPT1 substrates in the medium. This trans-stimulation phenomenon showed that hPEPT1 mediated the efflux of [3H]-Gly-Sar from CHO/hPEPT1 and that hPEPT1 is a bi-directional transporter. We then determined the effect of extracellular pH (varying from 8.0 to 3.5) on the efflux activity. The efflux activity by hPEPT1 decreased with the decrease in extracellular pH. The Henderson-Hasselbälch-type equation, which fitted well to the pH-profile of efflux activity, indicated that a single amino acid residue with a pKa value of approximately 5.7 regulates the efflux activity. The pH-profile of the efflux activity remained almost unchanged irrespective of the proton gradient across the plasma membrane. In addition, the chemical modification of the histidine residue with diethylpyrocarbonate completely abolished the efflux activity from cells, which could be prevented by the presence of 10 mM Gly-Sar. These data indicate that the efflux process of hPEPT1 is also regulated in a pH-dependent manner by the protonation state of a histidine residue located at or near the substrate recognition site facing the extracellular space.

Functional Mechanism of Cl--Pump Rhodopsin and Its Conversion into H+ Pump.

Cl--pump rhodopsin is the second discovered microbial rhodopsin. Although its physiological role has not been fully clarified, its functional mechanism has been studied as a model for anion transporters. After the success of neural activation by channel rhodopsin, the first Cl--pump halorhodopsin (HR) had become widely used as a neural silencer. The emergence of artificial and natural anion channel rhodopsins lowered the importance of HRs. However, the longer absorption maxima of approximately 585-600 nm for HRs are still advantageous for applications in mammalian brains and collaborations with neural activators possessing shorter absorption maxima. In this chapter, the variation and functional mechanisms of Cl- pumps are summarized. After the discovery of HR, Cl--pump rhodopsins were confined to only extremely halophilic haloarchaea. However, after 2014, two Cl--pump groups were newly discovered in marine and terrestrial bacteria. These Cl- pumps are phylogenetically distinct from HRs and have unique characteristics. In particular, the most recently identified Cl- pump has close similarity with the H+ pump bacteriorhodopsin and was converted into the H+ pump by a single amino acid replacement.

Protochromic absorption changes in the two-cysteine photocycle of a blue/orange cyanobacteriochrome.

Cyanobacteriochromes (CBCRs) are phytochrome-related photosensors with diverse spectral sensitivities spanning the entire visible spectrum. They covalently bind bilin chromophores via conserved cysteine residues and undergo 15Z/15E bilin photoisomerization upon light illumination. CBCR subfamilies absorbing violet-blue light use an additional cysteine residue to form a second bilin-thiol adduct in a two-Cys photocycle. However, the process of second thiol adduct formation is incompletely understood, especially the involvement of the bilin protonation state. Here, we focused on the Oscil6304_2705 protein from the cyanobacterium Oscillatoria acuminata PCC 6304, which photoconverts between a blue-absorbing 15Z state ( 15Z Pb) and orange-absorbing 15E state ( 15E Po). pH titration analysis revealed that 15Z Pb was stable over a wide pH range, suggesting that bilin protonation is stabilized by a second thiol adduct. As revealed by resonance Raman spectroscopy, 15E Po harbored protonated bilin at both acidic and neutral pH, but readily converted to a deprotonated green-absorbing 15Z state ( 15Z Pg) at alkaline pH. Site-directed mutagenesis revealed that the conserved Asp-71 and His-102 residues are required for second thiol adduct formation in 15Z Pb and bilin protonation in 15E Po, respectively. An Oscil6304_2705 variant lacking the second cysteine residue, Cys-73, photoconverted between deprotonated 15Z Pg and protonated 15E Pr, similarly to the protochromic photocycle of the green/red CBCR subfamily. Time-resolved spectroscopy revealed 15Z Pg formation as an intermediate in the 15E Pr-to- 15Z Pg conversion with a significant solvent-isotope effect, suggesting the sequential occurrence of 15EP-to-15Z photoisomerization, deprotonation, and second thiol adduct formation. Our findings uncover the details of protochromic absorption changes underlying the two-Cys photocycle of violet-blue-absorbing CBCR subfamilies.

Direct Detection of the Substrate Uptake and Release Reactions of the Light-Driven Sodium-Pump Rhodopsin.

For membrane transporters, substrate uptake and release reactions are major events during their transport cycles. Despite the functional importance of these events, it is difficult to identify their relevant structural intermediates because of the requirements of the experimental methods, which are to detect the timing of the formation and decay of intermediates and to detect the timing of substrate uptake and release. We report successfully achieving this for the light-driven Na+ pump rhodopsin (NaR). Here, a Na+-selective membrane, which consists of polyvinyl chloride and a Na+ ionophore, was employed to detect Na+ uptake and release. When one side of the membrane was covered by the lipid-reconstituted NaR, continuous illumination induced an increase in membrane potential, which reflected Na+ uptake by the photolyzed NaR. Via use of nanosecond laser pulses, two kinds of data were obtained during a single transport cycle: one was the flash-induced absorbance change in NaR to detect the formation and decay of structural intermediates, and the other was the flash-induced change in membrane potential, which reflects the transient Na+ uptake and release reactions. Their comparison clearly indicated that Na+ is captured and released during the formation and decay of the O intermediate, the red-shifted intermediate that appears in the latter half of the transport cycle.

Photoisomerization of azobenzene units drives the photochemical reaction cycles of proteorhodopsin and bacteriorhodopsin analogues.

In this study we substituted the retinal units in proteorhodopsin (PR) and bacteriorhodopsin (BR) with azo chromophores to investigate the mechanism of photoinduced proton pumping in rhodopsins and potentially develop new artificial molecular pumps. We used an indium tin oxide electrode to investigate the photoinduced proton transfer of the azo analogues of PR and BR. We also employed flash photolysis to determine the characteristic photocycles, comprising multiple transient intermediates, of the azo chromophore-bound PR and BR. Moreover, our studies of the photoinduced proton pumping functions of azo-proteoopsin and azo-bacterioopsin complexes revealed that they did not pump protons upon illumination, even though they underwent photoinduced proton transfer and the characteristic photocycle. Mutational analysis suggested that the proton pumping malfunction of the azo analogues of PR and BR resulted from the absence of proton transfer reactions through cytoplasmic channels, even though these reactions were evoked in extracellular channels. Based on our experimental findings, we propose herein a putative model of the proton transfer reaction mechanism for the azo analogues of PR and BR.

Low-Temperature Raman Spectroscopy of Halorhodopsin from Natronomonas pharaonis: Structural Discrimination of Blue-Shifted and Red-Shifted Photoproducts.

From the low-temperature absorption and Raman measurements of halorhodopsin from Natronomonas pharaonis (pHR), we observed that the two photoproducts were generated after exciting pHR at 80 K by green light. One photoproduct was the red-shifted K intermediate (pHRK) as the primary photointermediate for Cl- pumping, and the other was the blue-shifted one (pHRhypso), which was not involved in the Cl- pumping and thermally relaxed to the original unphotolyzed state by increasing temperature. The formation of these two kinds of photoproducts was previously reported for halorhodopsin from Halobacterium sarinarum [ Zimanyi et al. Biochemistry 1989 , 28 , 1656 ]. We found that the same took place in pHR, and we revealed the chromophore structures of the two photointermediates from their Raman spectra for the first time. pHRhypso had the distorted all-trans chromophore, while pHRK contained the distorted 13-cis form. The present results revealed that the structural analyses of pHRK carried out so far at ∼80 K potentially included a significant contribution from pHRhypso. pHRhypso was efficiently formed via the photoexcitation of pHRK, indicating that pHRhypso was likely a side product after photoexcitation of pHRK. The formation of pHRhypso suggested that the active site became tight in pHRK due to the slight movement of Cl-, and the back photoisomerization then produced the distorted all-trans chromophore in pHRhypso.

Spectroscopic characteristics of Rubricoccus marinus xenorhodopsin (RmXeR) and a putative model for its inward H+ transport mechanism.

A new group of microbial rhodopsins named xenorhodopsins (XeR), which are closely related to the cyanobacterial Anabaena sensory rhodopsin, show a light-driven "inward" proton transport activity, as reported for one representative of this group from Parvularcula oceani (PoXeR). In this study, we functionally and spectroscopically characterized a new member of the XeR clade from a marine bacterium Rubricoccus marinus SG-29T (RmXeR). Escherichia coli cells expressing recombinant RmXeR showed a light-induced alkalization of the cell suspension, which was strongly impaired by a protonophore, suggesting that RmXeR is a light-driven "inward" proton pump as is PoXeR. The spectroscopic properties of purified RmXeR were investigated and compared with those of PoXeR and a light-driven "outward" proton pump, bacteriorhodopsin (BR) from the archaeon Halobacterium salinarum. Action spectroscopy revealed that RmXeR with all-trans retinal is responsible for the light-driven inward proton transport activity, but not with 13-cis retinal. From pH titration experiments and mutational analysis, we estimated the pKa values for the protonated Schiff base of the retinal chromophore and its counterion as 11.1 ± 0.07 and 2.1 ± 0.07, respectively. Of note, the direction of both the retinal composition change upon light-dark adaptation and the acid-induced spectral shift was opposite that of BR, which is presumably related to the opposite directions of ion transport (from outside to inside for RmXeR and from inside to outside for BR). Flash photolysis experiments revealed the appearances of three intermediates (L, M and O) during the photocycle. The proton uptake and release were coincident with the formation and decay of the M intermediate, respectively. Together with associated findings from other microbial rhodopsins, we propose a putative model for the inward proton transport mechanism of RmXeR.

Characterization of a Cyanobacterial Chloride-pumping Rhodopsin and Its Conversion into a Proton Pump.

Light-driven ion-pumping rhodopsins are widely distributed in microorganisms and are now classified into the categories of outward H(+) and Na(+) pumps and an inward Cl(-) pump. These different types share a common protein architecture and utilize the photoisomerization of the same chromophore, retinal, to evoke photoreactions. Despite these similarities, successful pump-to-pump conversion had been confined to only the H(+) pump bacteriorhodopsin, which was converted to a Cl(-) pump in 1995 by a single amino acid replacement. In this study we report the first success of the reverse conversion from a Cl(-) pump to a H(+) pump. A novel microbial rhodopsin (MrHR) from the cyanobacterium Mastigocladopsis repens functions as a Cl(-) pump and belongs to a cluster that is far distant from the known Cl(-) pumps. With a single amino acid replacement, MrHR is converted to a H(+) pump in which dissociable residues function almost completely in the H(+) relay reactions. MrHR most likely evolved from a H(+) pump, but it has not yet been highly optimized into a mature Cl(-) pump.

Photochemical characterization of actinorhodopsin and its functional existence in the natural host.

Actinorhodopsin (ActR) is a light-driven outward H+ pump. Although the genes of ActRs are widely spread among freshwater bacterioplankton, there are no prior data on their functional expression in native cell membranes. Here, we demonstrate ActR phototrophy in the native actinobacterium. Genome analysis showed that Candidatus Rhodoluna planktonica, a freshwater actinobacterium, encodes one microbial rhodopsin (RpActR) belonging to the ActR family. Reflecting the functional expression of RpActR, illumination induced the acidification of the actinobacterial cell suspension and then elevated the ATP content inside the cells. The photochemistry of RpActR was also examined using heterologously expressed RpActR in Escherichia coli membranes. The purified RpActR showed λmax at 534nm and underwent a photocycle characterized by the very fast formation of M intermediate. The subsequent intermediate, named P620, could be assigned to the O intermediate in other H+ pumps. In contrast to conventional O, the accumulation of P620 remains prominent, even at high pH. Flash-induced absorbance changes suggested that there exists only one kind of photocycle at any pH. However, above pH7, RpActR shows heterogeneity in the H+ transfer sequences: one first captures H+ and then releases it during the formation and decay of P620, while the other first releases H+ prior to H+ uptake during P620 formation.

Formation of M-Like Intermediates in Proteorhodopsin in Alkali Solutions (pH ≥ ∼8.5) Where the Proton Release Occurs First in Contrast to the Sequence at Lower pH.

Proteorhodopsin (PR) is an outward light-driven proton pump observed in marine eubacteria. Despite many structural and functional similarities to bacteriorhodopsin (BR) in archaea, which also acts as an outward proton pump, the mechanism of the photoinduced proton release and uptake is different between two H(+)-pumps. In this study, we investigated the pH dependence of the photocycle and proton transfer in PR reconstituted with the phospholipid membrane under alkaline conditions. Under these conditions, as the medium pH increased, a blue-shifted photoproduct (defined as Ma), which is different from M, with a pKa of ca. 9.2 was produced. The sequence of the photoinduced proton uptake and release during the photocycle was inverted with the increase in pH. A pKa value of ca. 9.5 was estimated for this inversion and was in good agreement with the pKa value of the formation of Ma (∼ 9.2). In addition, we measured the photoelectric current generated by PRs attached to a thin polymer film at varying pH. Interestingly, increases in the medium pH evoked bidirectional photocurrents, which may imply a possible reversal of the direction of the proton movement at alkaline pH. On the basis of these findings, a putative photocycle and proton transfer scheme in PR under alkaline pH conditions was proposed.