The microbial opsin family of optogenetic tools.

The capture and utilization of light is an exquisitely evolved process. The single-component microbial opsins, although more limited than multicomponent cascades in processing, display unparalleled compactness and speed. Recent advances in understanding microbial opsins have been driven by molecular engineering for optogenetics and by comparative genomics. Here we provide a Primer on these light-activated ion channels and pumps, describe a group of opsins bridging prior categories, and explore the convergence of molecular engineering and genomic discovery for the utilization and understanding of these remarkable molecular machines.

Crystal structure of heliorhodopsin.

Heliorhodopsins (HeRs) are a family of rhodopsins that was recently discovered using functional metagenomics1. They are widely present in bacteria, archaea, algae and algal viruses2,3. Although HeRs have seven predicted transmembrane helices and an all-trans retinal chromophore as in the type-1 (microbial) rhodopsin, they display less than 15% sequence identity with type-1 and type-2 (animal) rhodopsins. HeRs also exhibit the reverse orientation in the membrane compared with the other rhodopsins. Owing to the lack of structural information, little is known about the overall fold and the photoactivation mechanism of HeRs. Here we present the 2.4-Å-resolution structure of HeR from an uncultured Thermoplasmatales archaeon SG8-52-1 (GenBank sequence ID LSSD01000000). Structural and biophysical analyses reveal the similarities and differences between HeRs and type-1 microbial rhodopsins. The overall fold of HeR is similar to that of bacteriorhodopsin. A linear hydrophobic pocket in HeR accommodates a retinal configuration and isomerization as in the type-1 rhodopsin, although most of the residues constituting the pocket are divergent. Hydrophobic residues fill the space in the extracellular half of HeR, preventing the permeation of protons and ions. The structure reveals an unexpected lateral fenestration above the ß-ionone ring of the retinal chromophore, which has a critical role in capturing retinal from environment sources. Our study increases the understanding of the functions of HeRs, and the structural similarity and diversity among the microbial rhodopsins.

A distinct abundant group of microbial rhodopsins discovered using functional metagenomics.

Many organisms capture or sense sunlight using rhodopsin pigments1,2, which are integral membrane proteins that bind retinal chromophores. Rhodopsins comprise two distinct protein families 1 , 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 helix2,3. 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 1 . 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.

A Novel Color Switch of Microbial Rhodopsin.

Function of animal and microbial rhodopsins starts by light absorption of the retinal chromophore. The absorption maximum wavelength (λmax) of rhodopsins is determined by the energy gap between the electronically ground (S0) and first excited (S1) state of the retinal chromophore, and the color tuning mechanism is one of the central topics in rhodopsin research. "Color switches", color-determining residues, are red- and blue-shifting amino acids at the same position in two rhodopsins, whose exchange causes spectral blue- and red-shifts, respectively, in each rhodopsin. As mutation easily destroys elaborate chromophore-protein interactions, the known color switches in microbial rhodopsins are limited; the L/Q switch in C-helix (TM3), the A/TS switch in G-helix (TM7), and the G/P switch in F-helix (TM6). Here, we report a novel color switch of microbial rhodopsins, which is located in D-helix (TM4). In this color switch, the red- and blue-shifting amino acids are Asn (N) and Leu (L)/Ile (I), respectively. As Asn and Leu/Ile are polar and nonpolar amino acids, respectively, and the position is located near the ß-ionone ring, the N/LI switch matches the general rule of color tuning by polarity. The N/LI switch is also useful for optogenetics, as many ion-transporting rhodopsins contain blue-shifting amino acids, such as L and I, at that position.

Optogenetic reprogramming of carbon metabolism using light-powering microbial proton pump systems.

In microbial fermentative production, ATP regeneration, while crucial for cellular processes, conflicts with efficient target chemical production because ATP regeneration exhausts essential carbon sources also required for target chemical biosynthesis. To wrestle with this dilemma, we harnessed the power of microbial rhodopsins with light-driven proton pumping activity to supplement with ATP, thereby facilitating the bioproduction of various chemicals. We first demonstrated a photo-driven ATP supply and redistribution of metabolic carbon flows to target chemical synthesis by installing already-known delta rhodopsin (dR) in Escherichia coli. In addition, we identified novel rhodopsins with higher proton pumping activities than dR, and created an engineered cell for in vivo self-supply of the rhodopsin-activator, all-trans-retinal. Our concept exploiting the light-powering ATP supplier offers a potential increase in carbon use efficiency for microbial productions through metabolic reprogramming.

Factors related to daily step counts of stroke patients during hospitalization in a convalescent rehabilitation ward.

OBJECTIVES: Clarifying the factors related to decreased physical activity in post-stroke patients is essential for effective disease management. This study aimed to examine the factors influencing the amount of daily steps taken by post-stroke patients in a convalescent rehabilitation ward during activities other than rehabilitation (non-rehabilitation steps). MATERIALS AND METHODS: Eighty-nine post-stroke patients (60.8±14.4 years; 55 men) were enrolled. The inclusion criteria were walking independently within the ward and having a walking speed of ≥24 m/min. Data on patient clinical characteristics including age, sex, body mass index, stroke type, hemiparetic side, and time from stroke onset were collected. Stroke impairment and motor and cognitive functional disabilities were assessed using the Stroke Impairment Assessment Set and the Functional Independence Measure, respectively. The non-rehabilitation steps were calculated by subtracting the steps during the rehabilitation activities from the total steps using Fitbit Flex2. RESULTS: The average number of non-rehabilitation steps was 4,523±2,339 steps/day. The hierarchical multiple regression analysis revealed that sex, motor disability, and the interaction term of stroke impairment with cognitive disability were significantly related to non-rehabilitation steps. Simple slope analysis demonstrated that the stroke impairment slope was steeper at lower levels than at higher levels of cognitive disability for non-rehabilitation steps. CONCLUSIONS: In addition to independent effects of sex and motor disability, this study found that stroke impairment and cognitive disability were interactively related to non-rehabilitation steps in post-stroke patients in a convalescent rehabilitation ward. These findings may provide useful information for managing physical activity in post-stroke patients after hospital discharge.

TAT Rhodopsin Is an Ultraviolet-Dependent Environmental pH Sensor.

In many rhodopsins, the retinal Schiff base pKa remains very high, ensuring Schiff base protonation captures visible light. Nevertheless, recently we found that TAT rhodopsin contains protonated and unprotonated forms at physiological pH. The protonated form displays a unique photochemical behavior in which the primary K intermediate returns to the original state within 10-5 s, and the lack of photocycle completion poses questions about the functional role of TAT rhodopsin. Here we studied the molecular properties of the protonated and unprotonated forms of the Schiff base in TAT rhodopsin. We confirmed no photointermediate formation at >10-5 s for the protonated form of TAT rhodopsin in microenvironments such as detergents, nanodiscs, and liposomes. In contrast, the unprotonated form features a very long photocycle with a time constant of 15 s. A low-temperature study revealed that the primary reaction of the unprotonated form is all-trans to 13-cis photoisomerization, which is usual, but with a proton transfer reaction occurring at 77 K, which is unusual. The active intermediate contains the unprotonated Schiff base as well as the resting state. Electrophysiological measurements excluded ion-transport activity for TAT rhodopsin, while transient outward proton movement only at an alkaline extracellular pH indicates that TAT rhodopsin senses the extracellular pH. On the basis of the findings presented here, we propose that TAT rhodopsin is an ultraviolet (UV)-dependent environmental pH sensor in marine bacteria. At acidic pH, absorbed visible light energy is quickly dissipated into heat without any function. In contrast, when the environmental pH becomes high, absorption of UV/blue light yields formation of the long-lived intermediates, possibly driving the signal transduction cascade in marine bacteria.

Molecular Properties and Optogenetic Applications of Enzymerhodopsins.

The cyclic nucleotides cAMP and cGMP are ubiquitous secondary messengers that regulate multiple biological functions including gene expression, differentiation, proliferation, and cell survival. In sensory neurons, cyclic nucleotides are responsible for signal modulation, amplification, and encoding. For spatial and temporal manipulation of cyclic nucleotide dynamics, optogenetics have a great advantage over pharmacological approaches. Enzymerhodopsins are a unique family of microbial rhodopsins. These molecules are made up of a membrane-embedded rhodopsin domain, which binds an all trans-retinal to form a chromophore, and a cytoplasmic water-soluble catalytic domain. To date, three kinds of molecules have been identified from lower eukaryotes such as fungi, algae, and flagellates. Among these, histidine kinase rhodopsin (HKR) is a light-inhibited guanylyl cyclase. Rhodopsin GC (Rh-GC) functions as a light-activated guanylyl cyclase, while rhodopsin PDE (Rh-PDE) functions as a light-activated phosphodiesterase that degrades cAMP and cGMP. These enzymerhodopsins have great potential in optogenetic applications for manipulating the intracellular cyclic nucleotide dynamics of living cells. Here we introduce the molecular function and applicability of these molecules.

Spectroscopic study of the transmembrane domain of a rhodopsin-phosphodiesterase fusion protein from a unicellular eukaryote.

The choanoflagellate Salpingoeca rosetta contains a chimeric rhodopsin protein composed of an N-terminal rhodopsin (Rh) domain and a C-terminal cyclic nucleotide phosphodiesterase (PDE) domain. The Rh-PDE enzyme light-dependently decreases the concentrations of cyclic nucleotides such as cGMP and cAMP. Photoexcitation of purified full-length Rh-PDE yields an "M" intermediate with a deprotonated Schiff base, and its recovery is much faster than that of the enzyme domain. To gain structural and mechanistic insights into the Rh domain, here we expressed and purified the transmembrane domain of Rh-PDE, Rh-PDE(TMD), and analyzed it with transient absorption, light-induced difference UV-visible, and FTIR spectroscopy methods. These analyses revealed that the "K" intermediate forms within 0.005 ms and converts into the M intermediate with a time constant of 4 ms, with the latter returning to the original state within 4 s. FTIR spectroscopy revealed that all-trans to 13-cis photoisomerization occurs as the primary event during which chromophore distortion is located at the middle of the polyene chain, allowing the Schiff base to form a stronger hydrogen bond. We also noted that the peptide backbone of the α-helix becomes deformed upon M intermediate formation. Results from site-directed mutagenesis suggested that Glu-164 is protonated and that Asp-292 acts as the only Schiff base counterion in Rh-PDE. A strong reduction of enzymatic activity in a D292N variant, but not in an E164Q variant, indicated an important catalytic role of the negative charge at Asp-292. Our findings provide further mechanistic insights into rhodopsin-mediated, light-dependent regulation of second-messenger levels in eukaryotic microbes.

Light-Driven Sodium-Pumping Rhodopsin: A New Concept of Active Transport.

Ion pumps perform active transport of ions by using energy. The active transport mechanism can be illustrated by the Panama Canal model, which considers two gates and a gain in energy. The Panama Canal model is consistent with the alternating access model that is used to describe active transport, in which the substrate ion is bound, energized, and released. It was generally accepted that energization occurs only for an ion-bound protein but not for an ion-unbound protein. Light-driven proton and chloride pumps, two of the best studied pumps, are represented by the Panama Canal model. In this case, light absorption takes place for the bound state of ions (proton and chloride ions) in the active center (protonated Schiff base of the retinal chromophore). In contrast, a recently discovered light-driven sodium pump, Krokinobacter eikastus rhodopsin 2 (KR2), is a unique active transporter that does not bind the transport substrate, the sodium ion, in its resting state. The molecular architecture and photoreaction cycle of the light-driven sodium pump are very similar to those of proton and chloride pumps, although sodium ions are actively transported without initial binding. Sodium uptake is a diffusive process, but the presence of two gates allows the unidirectional transport of sodium ions. In this sense, the light-driven sodium pump is also represented by a modified Panama Canal model. Current understanding of the light-driven sodium pump is reviewed.

A collagen-coated PGA conduit for interpositional-jump graft with end-to-side neurorrhaphy for treating facial nerve paralysis in rat.

PURPOSE: This study investigated the potential of collagen-coated polyglycolic acid (PGA) tube with interpositional jump graft (IPJG) in rat. MATERIALS AND METHODS: A total of 16 Lewis rats were used in this study. Facial nerve paralysis was created by ligating facial nerve trunk with a ligature clip. The rats were divided into 3 groups. Nerve conduit group (n = 6) were treated by IPJG with collagen-coated PGA tubes between the facial nerve trunks and the hypoglossal nerves. Autograft group (n = 6) were treated by IPJG with the greater auricular nerves. As the control group (n = 4), non-treated-model rats with facial nerve paralysis were used. The number of myelinated fibers, fiber diameter, axon diameter, myelin thickness, and g-ratio, were analyzed histologically at 13 weeks after surgery. Compound muscle action potential (CMAP) and retrograde tracing were measured. RESULT: Although the number of myelinated fibers in autograft group (1957 ± 775) had significantly higher than that of nerve conduit group (90 ± 41, P < .05), the nerve conduit group showed the regeneration of myelinated nerve axons. CMAP amplitude values of the autograft (4706 ± 1154 µV) and the nerve conduit groups (4119 ± 1397 µV) were significantly higher than that of the control group (915 ± 789 µV, P < .05). Retrograde tracing confirmed the double innervation of mimetic muscles by the facial and hypoglossal nucleus in both groups. CONCLUSION: This study showed histologically and physiologically the superior effectiveness of performing IPJG with a collagen-coated PGA conduit in a rat model.

A unique choanoflagellate enzyme rhodopsin exhibits light-dependent cyclic nucleotide phosphodiesterase activity.

Photoactivated adenylyl cyclase (PAC) and guanylyl cyclase rhodopsin increase the concentrations of intracellular cyclic nucleotides upon illumination, serving as promising second-generation tools in optogenetics. To broaden the arsenal of such tools, it is desirable to have light-activatable enzymes that can decrease cyclic nucleotide concentrations in cells. Here, we report on an unusual microbial rhodopsin that may be able to meet the demand. It is found in the choanoflagellate Salpingoeca rosetta and contains a C-terminal cyclic nucleotide phosphodiesterase (PDE) domain. We examined the enzymatic activity of the protein (named Rh-PDE) both in HEK293 membranes and whole cells. Although Rh-PDE was constitutively active in the dark, illumination increased its hydrolytic activity 1.4-fold toward cGMP and 1.6-fold toward cAMP, as measured in isolated crude membranes. Purified full-length Rh-PDE displayed maximal light absorption at 492 nm and formed the M intermediate with the deprotonated Schiff base upon illumination. The M state decayed to the parent spectral state in 7 s, producing long-lasting activation of the enzyme domain with increased activity. We discuss a possible mechanism of the Rh-PDE activation by light. Furthermore, Rh-PDE decreased cAMP concentration in HEK293 cells in a light-dependent manner and could do so repeatedly without losing activity. Thus, Rh-PDE may hold promise as a potential optogenetic tool for light control of intracellular cyclic nucleotides (e.g. to study cyclic nucleotide-associated signal transduction cascades).

Adipose-derived stem cells and the stromal vascular fraction in polyglycolic acid-collagen nerve conduits promote rat facial nerve regeneration.

Adipose-derived stem cells (ADSCs) and the stromal vascular fraction (SVF) promote nerve regeneration. Biodegradable nerve conduits are used to treat peripheral nerve injuries, but their efficiencies are lower than those of autologous nerve grafts. This study developed biodegradable nerve conduits containing ADSCs and SVF and evaluated their facial nerve regenerating abilities in a rat model with a 7-mm nerve defect. SVF and ADSCs were individually poured into nerve conduits with polyglycolic acid-type I collagen as a scaffold (ADSCs and SVF groups). The conduits were grafted on to the nerve defects. As the control, the defect was bridged with polyglycolic acid-collagen nerve conduits without cells. At 13 weeks, after transplantation, the regenerated nerves were evaluated physiologically and histologically. The compound muscle action potential of the SVF group was significantly higher in amplitude than that of the control group. Electron microscopy showed that the axon diameter of the SVF group was the largest, followed by the ADSC group and control group with significant differences among them. The SVF group had the largest fiber diameter, followed by the ADSC group and control group with significant differences among them. The ADSC group had the highest myelin thickness, followed by the SVF group and control group with significant differences among them. Identical excellent promoting effects on nerve regeneration were observed in both the ADSC and SVF groups. Using SVF in conduits was more practical than using ADSCs because only the enzymatic process was required to prepare SVF, indicating that SVF could be more suitable to induce nerve regeneration.

Axonal supercharged interpositional jump-graft with a hybrid artificial nerve conduit containing adipose-derived stem cells in facial nerve paresis rat model.

PURPOSE: Interpositional jump-graft (IPJG) technique with the hypoglossal nerve for supercharging can be applied in a facial nerve paresis case. In IPJG, an autologous nerve is required, and the donor site morbidity is unavoidable. Biodegradable nerve conduits are made from polyglycolic acid (PGA) and used recently without donor site complications after providing autologous grafts. Hybrid artificial nerve conduits with adipose-derived stem cells (ASCs) also attract attention as a nerve-regeneration enhancing agent. This study examined the effect of hybrid artificial nerve conduit on IPJG. MATERIALS AND METHODS: A total of 34 Lewis rats were used and divided into 4 groups by the bridge materials: autograft (n = 8), PGA nerve conduit (n = 8), hybrid PGA nerve conduit with ASCs (n = 8), and the nontreated control groups (n = 8). ASCs were collected from 2 rats and cultured. The animals were assessed physiologically and histopathologically at 13 weeks after surgery. RESULTS: In compound muscle action potential, the amplitude of hybrid PGA group (3,222 ± 1,779 µV) was significantly higher than that of PGA group (1,961 ± 445 µV, P < .05), and no significant difference between hybrid PGA and autograft group. All treated groups showed a myelinated nerve regeneration with double innervation in hypoglossal and facial nerve nuclei for vibrissal muscle. CONCLUSION: This study showed the effectiveness of IPJG with a hybrid PGA conduit especially in physiological examination.

Adipose-derived aldehyde dehydrogenase-expressing cells promote dermal regenerative potential with collagen-glycosaminoglycan scaffold.

Aldehyde dehydrogenase (ALDH) is an enzyme that plays an important role in retinoid metabolism and highly expressed in stem cells. This study isolated ALDH-expressing cells from subcutaneous adipose tissue and investigated their potential to enhance healing in a full-thickness skin wound in rats by co-implanting them with collagen-glycosaminoglycan (c-GAG) scaffolds. ALDH-positive cells were isolated by a fluorescence-activated cell sorting technique from Lewis rat's stromal-vascular-fraction (SVF) and transplanted with c-GAG scaffolds in a rat full-thickness skin wound model. At 7 days after surgery, the microscopic appearance of c-GAG scaffolds seeded with ALDH-positive was compared with those of uncultured-SVF, and cultured-SVF adipose-derived stromal cells (ASCs). The thickness of cellular ingrowth in the ASC group (630 ± 180 µm) was significantly thicker than that in the control (390 ± 120 µm) or SVF (380 ± 140 µm) groups, but non-significantly thicker than that in the ALDH-positive group (570 ± 220 µm). The thickness of regenerated collagen layer was significantly thicker in the ALDH-positive group (160 ± 110 µm) than in the ASCs (81 ± 41 µm), the control (65 ± 24 µm), or SVF (64 ± 34 µm) groups. Immunofluorescent staining with CD31 proved that transplanted ALDH-positive cells differentiated into vascular endothelial cells in c-GAG scaffolds. Combined transplantation with c-GAG scaffolds and adipose-derived ALDH-positive cells promoted dermal regeneration, giving a possibility that ALDH-positive cells would greatly shorten the waiting period before secondary autologous skin grafting was possible.

Oxidative stress and redox regulation of gametogenesis, fertilization, and embryonic development.

Oxidative stress caused by elevated reactive oxygen species (ROS) is one of the predominant causes of both male and female infertility. Oxidative stress conditions cause either cell death or senescence by oxidation of cellular molecules including nucleic acid, proteins, and lipids. It is particularly important to minimize oxidative stress when in vitro fertilization is performed for the purpose of assisted reproduction. The problems associated with assisted reproductive technology are becoming evident, and it is now the time to clarify its mechanisms and cope with them. On the other hand, the beneficial roles of ROS, such as intracellular signaling, have become evident. The antithetical functions of ROS make it more difficult to overcome the problems caused by oxidative stress. Despite the difficulty in understanding mammalian reproduction, the mechanisms and problems can be gradually unveiled by advanced technology such as genetic modification of animals.

Structural model of channelrhodopsin.

Channelrhodopsins (ChRs) are light-gated cation channels that mediate ion transport across membranes in microalgae (vectorial catalysis). ChRs are now widely used for the analysis of neural networks in tissues and living animals with light (optogenetics). For elucidation of functional mechanisms at the atomic level, as well as for further engineering and application, a detailed structure is urgently needed. In the absence of an experimental structure, here we develop a structural ChR model based on several molecular computational approaches, capitalizing on characteristic patterns in amino acid sequences of ChR1, ChR2, Volvox ChRs, Mesostigma ChR, and the recently identified ChR of the halophilic alga Dunaliella salina. In the present model, we identify remarkable structural motifs that may explain fundamental electrophysiological properties of ChR2, ChR1, and their mutants, and in a crucial validation of the model, we successfully reproduce the excitation energy predicted by absorption spectra.

Color-tuned channelrhodopsins for multiwavelength optogenetics.

Channelrhodopsin-2 is a light-gated ion channel and a major tool of optogenetics. It is used to control neuronal activity via blue light. Here we describe the construction of color-tuned high efficiency channelrhodopsins (ChRs), based on chimeras of Chlamydomonas channelrhodopsin-1 and Volvox channelrhodopsin-1. These variants show superb expression and plasma membrane integration, resulting in 3-fold larger photocurrents in HEK cells compared with channelrhodopsin-2. Further molecular engineering gave rise to chimeric variants with absorption maxima ranging from 526 to 545 nm, dovetailing well with maxima of channelrhodopsin-2 derivatives ranging from 461 to 492 nm. Additional kinetic fine-tuning led to derivatives in which the lifetimes of the open state range from 19 ms to 5 s. Finally, combining green- with blue-absorbing variants allowed independent activation of two distinct neural cell populations at 560 and 405 nm. This novel panel of channelrhodopsin variants may serve as an important toolkit element for dual-color cell stimulation in neural circuits.

Differential responses of SOD1-deficient mouse embryonic fibroblasts to oxygen concentrations.

Superoxide dismutase (SOD) plays a role in antioxidation, and SOD1-knockout (KO) mice show moderate phenotypes. Primary cultured mouse embryonic fibroblasts (MEFs) lead to growth failure and eventual death under normoxic culture (20% oxygen). We attempted to elucidate the molecular mechanisms responsible for the oxygen toxicity in SOD1-KO MEFs. Increases in reactive oxygen species, lipid peroxidation products, and senescence-associated ß-galactosidase activity were observed in SOD1-KO MEFs. Hypoxic culture (2% oxygen) averted immediate cell death but could not recover the proliferative ability of the SOD1-KO cells. The cell cycles of SOD1-deficient MEFs were arrested at the G2 and M phases, leading to the accumulation of tetraploid cells under hypoxic culture. The suppressed expression of cyclin A2 and B1 and the concomitant induction of p21(Waf1) were evident in SOD1-KO cells. The phosphorylation of p53 and histone H2Ax and the induction of the two proapoptotic genes Bax and Noxa were evident in SOD1-deficient MEFs and more enhanced under normoxic culture than under hypoxic culture. We concluded that low levels of oxygen consumption moderately activates the p53 pathway, and leads to cellular senescence, but that high levels of oxygen consumption hyperactivates the p53 pathway, which results in cell death in SOD1-deficient MEFs.

Light-Driven Chloride and Sulfate Pump with Two Different Transport Modes.

Ion pumps are membrane proteins that actively translocate ions by using energy. All known pumps bind ions in the resting state, and external energy allows ion transport through protein structural changes. The light-driven sodium-ion pump Krokinobacter eikastus rhodopsin 2 (KR2) is an exceptional case in which ion binding follows the energy input. In this study, we report another case of this unusual transport mode. The NTQ rhodopsin from Alteribacter aurantiacus (AaClR) is a natural light-driven chloride pump, in which the chloride ion binds to the resting state. AaClR is also able to pump sulfate ions, though the pump efficiency is much lower for sulfate ions than for chloride ions. Detailed spectroscopic analysis revealed no binding of the sulfate ion to the resting state of AaClR, indicating that binding of the substrate (sulfate ion) to the resting state is not necessary for active transport. This property of the AaClR sulfate pump is similar to that of the KR2 sodium pump. Photocycle dynamics of the AaClR sulfate pump resemble a non-functional cycle in the absence of anions. Despite this, flash photolysis and difference Fourier transform infrared spectroscopy suggest transient binding of the sulfate ion to AaClR. The molecular mechanism of this unusual active transport by AaClR is discussed.