A Proteorhodopsin-Related Photosensor Expands the Repertoire of Structural Motifs Employed by Sensory Rhodopsins.

Microbial rhodopsins are light-activated retinal-binding membrane proteins that perform a variety of ion transport and photosensory functions. They display several cases of convergent evolution where the same function is present in unrelated or very distant protein groups. Here we report another possible case of such convergent evolution, describing the biophysical properties of a new group of sensory rhodopsins. The first representative of this group was identified in 2004 but none of the members had been expressed and characterized. The well-studied haloarchaeal sensory rhodopsins interacting with methyl-accepting Htr transducers are close relatives of the halobacterial proton pump bacteriorhodopsin. In contrast, the sensory rhodopsins we describe here are relatives of proteobacterial proton pumps, proteorhodopsins, but appear to interact with Htr-like transducers likewise, even though they do not conserve the residues important for the interaction of haloarchaeal sensory rhodopsins with their transducers. The new sensory rhodopsins display many unusual amino acid residues, including those around the retinal chromophore; most strikingly, a tyrosine in place of a carboxyl counterion of the retinal Schiff base on helix C. To characterize their unique sequence motifs, we augment the spectroscopy and biochemistry data by structural modeling of the wild-type and three mutants. Taken together, the experimental data, bioinformatics sequence analyses, and structural modeling suggest that the tyrosine/aspartate complex counterion contributes to a complex water-mediated hydrogen-bonding network that couples the protonated retinal Schiff base to an extracellular carboxylic dyad.

Key determinants for signaling in the sensory rhodopsin II/transducer complex are different between Halobacterium salinarum and Natronomonas pharaonis.

The cell membrane of Halobacterium salinarum contains a retinal-binding photoreceptor, sensory rhodopsin II (HsSRII), coupled with its cognate transducer (HsHtrII), allowing repellent phototaxis behavior for shorter wavelength light. Previous studies on SRII from Natronomonas pharaonis (NpSRII) pointed out the importance of the hydrogen bonding interaction between Thr204NpSRII and Tyr174NpSRII in signal transfer from SRII to HtrII. Here, we investigated the effect on phototactic function by replacing residues in HsSRII corresponding to Thr204NpSRII and Tyr174NpSRII . Whereas replacement of either residue altered the photocycle kinetics, introduction of any mutations at Ser201HsSRII and Tyr171HsSRII did not eliminate negative phototaxis function. These observations imply the possibility of the presence of an unidentified molecular mechanism for photophobic signal transduction differing from NpSRII-NpHtrII.

Pathogenic STX3 variants affecting the retinal and intestinal transcripts cause an early-onset severe retinal dystrophy in microvillus inclusion disease subjects.

Biallelic STX3 variants were previously reported in five individuals with the severe congenital enteropathy, microvillus inclusion disease (MVID). Here, we provide a significant extension of the phenotypic spectrum caused by STX3 variants. We report ten individuals of diverse geographic origin with biallelic STX3 loss-of-function variants, identified through exome sequencing, single-nucleotide polymorphism array-based homozygosity mapping, and international collaboration. The evaluated individuals all presented with MVID. Eight individuals also displayed early-onset severe retinal dystrophy, i.e., syndromic-intestinal and retinal-disease. These individuals harbored STX3 variants that affected both the retinal and intestinal STX3 transcripts, whereas STX3 variants affected only the intestinal transcript in individuals with solitary MVID. That STX3 is essential for retinal photoreceptor survival was confirmed by the creation of a rod photoreceptor-specific STX3 knockout mouse model which revealed a time-dependent reduction in the number of rod photoreceptors, thinning of the outer nuclear layer, and the eventual loss of both rod and cone photoreceptors. Together, our results provide a link between STX3 loss-of-function variants and a human retinal dystrophy. Depending on the genomic site of a human loss-of-function STX3 variant, it can cause MVID, the novel intestinal-retinal syndrome reported here or, hypothetically, an isolated retinal dystrophy.

The SNARE regulator Complexin3 is a target of the cone circadian clock.

BMAL1 is a core component of the mammalian circadian clockwork. Removal of BMAL1 from the retina significantly affects visual information processing in both rod and cone pathways. To identify potential pathways and/or molecules through which BMAL1 alters signal transmission at the cone pedicle, we performed an RNA-seq differential expression analysis between cone-specific Bmal1 knockout cones (cone-Bmal1-/- ) and wild-type (WT) cones. We found 88 genes differentially expressed. Among these, Complexin3 (Cplx3), a SNARE regulator at ribbon synapses, was downregulated fivefold in the mutant cones. The purpose of this work was to determine whether BMAL1 and/or the cone clock controls CPLX3 protein expression at cone pedicles. We found that CPLX3 expression level was decreased twofold in cone-Bmal1-/- cones. Furthermore, CPLX3 expression was downregulated at night compared to the day in WT cones but remained constitutively low in mutant cones both day and night. The transcript and protein expression levels of Cplx4-the other complexin expressed in cones-were similar in WT and mutant cones; in WT cones, CPLX4 protein level did not change with the time of day. In silico analysis revealed four potential BMAL1:CLOCK binding sites upstream from exon one of Cplx3 and none upstream of exon one of Cplx4. Our results suggest that CPLX3 expression is regulated at the transcriptional level by the cone clock. The modulation of CPLX3 may be a mechanism by which the clock controls the cone synaptic transfer function to second-order cells and thereby impacts retinal signal processing during the day/night cycle.

Parcas is the predominant Rab11-GEF for rhodopsin transport in Drosophila photoreceptors.

Rab11 is essential for polarized post-Golgi vesicle trafficking to photosensitive membrane rhabdomeres in Drosophila photoreceptors. Here, we found that Parcas (Pcs), recently shown to have guanine nucleotide exchange (GEF) activity toward Rab11, co-localizes with Rab11 on the trans-side of Golgi units and post-Golgi vesicles at the base of the rhabdomeres in pupal photoreceptors. Pcs fused with the electron micrography tag APEX2 localizes on 150-300 nm vesicles at the trans-side of Golgi units, which are presumably fly recycling endosomes. Loss of Pcs impairs Rab11 localization on the trans-side of Golgi units and induces the cytoplasmic accumulation of post-Golgi vesicles bearing rhabdomere proteins, as observed in Rab11 deficiency. In contrast, loss of Rab11-specific subunits of the TRAPPII complex, another known Rab11-GEF, does not cause any defects in eye development nor the transport of rhabdomere proteins; however, simultaneous loss of TRAPPII and Pcs results in severe defects in eye development. These results indicate that both TRAPPII and Pcs are required for eye development, but Pcs functions as the predominant Rab11-GEF for post-Golgi transport to photosensitive membrane rhabdomeres.

Establishment of an induced pluripotent stem cell line (FRIMOi005-A) derived from a retinitis pigmentosa patient carrying a dominant mutation in RHO gene.

Retinitis pigmentosa (RP) is an inherited retinal dystrophy characterized by the progressive degeneration of photoreceptors. In the present study, we have generated an induced pluripotent stem cell (iPSC) line derived from a RP patient with a dominant mutation in the RHO gene, responsible for the synthesis of rhodopsin. The reprogramming of these iPSCs was performed from skin fibroblasts by the Sendai-virus based approach. Characterization of the iPSC line showed a normal karyotype carrying the RHO mutation, expressed pluripotency markers and could be differentiated to endoderm, mesoderm and ectoderm in vitro.

Point Mutation of Anabaena Sensory Rhodopsin Enhances Ground-State Hydrogen Out-of-Plane Wag Raman Activity.

The interaction between the retinal protonated Schiff base (RPSB) and surrounding protein residues inside the retinal pocket is believed to play a major role in the ultrafast isomerization of the former. Coherent time-resolved vibrational spectroscopic techniques are applied to reveal the effect of changes in the protein architecture by point mutations (V112N and L83Q) close to the RPSB in Anabaena sensory rhodopsin (ASR). Our study reveals that such point mutations have a minor effect on the low-frequency (<400 cm-1) torsional modes but dramatically influence the ground-state vibrational Raman activity of the C14-H out-of-plane (HOOP) wag mode (800-820 cm-1). In mutated ASR, the increase of HOOP Raman activity in the ground state is experimentally observed for the all- trans RPSB, which has shorter excited-state lifetime than in wild-type ASR. This indicates that predistortion of the RPSB inside the mutated retinal pocket is a major factor in the acceleration of the isomerization rate.

Heliorhodopsins are absent in diderm (Gram-negative) bacteria: Some thoughts and possible implications for activity.

Microbial heliorhodopsins are a new type of rhodopsins, currently believed to engage in light sensing, with an opposite membrane topology compared to type-1 and type-2 rhodopsins. We determined heliorhodopsins presence/absence is monoderms and diderms representatives from the Tara Oceans and freshwater metagenomes as well as metagenome assembled genome collections. Heliorhodopsins are absent in diderms, confirming our previous observations in cultured Proteobacteria. We do not rule out the possibility that heliorhodopsins serve as light sensors. However, this does not easily explain their absence from diderms. Based on these observations, we speculate on the putative role of heliorhodopsins in light-driven transport of amphiphilic molecules.

Expression of Anabaena sensory rhodopsin is influenced by different codons of seven residues at the N-terminal region.

Microbial rhodopsins are well-known seven-transmembrane proteins that have been extensively studied for their structure and function. These retinal-binding proteins can be divided into two types. Type I is microbial rhodopsin, and type II (visual pigment) is expressed mostly in mammalian eyes. The two primary functions of type I rhodopsin are ion pumping activity and sensory transduction. Anabaena sensory rhodopsin (ASR) is a microbial rhodopsin with a specific function of photosensory transduction. ASR is expressed at moderate levels in Escherichia coli, but its expression level is lower compared to the general green light absorbing proteorhodopsin (GPR). In this study, full-length ASR was used to test the influence of codon usage on expression E. coli. Seven amino acids at the N-terminal region of ASR after the Met start codon were changed randomly using designed primers, which allowed for 8192 different nucleotide combinations. The codon changes were screened for the preferable codons that resulted in higher expression yield. Among the 57 selected mutations, 24 color-enhanced E. coli colonies contained ASR proteins, and they expressed ASR at a higher level than the bacteria with wild-type ASR codon usage. This result strongly suggests that the specific codon usage of only the N-terminal portion of a protein can increase the expression level of the entire protein.

Effect of point mutations on the ultrafast photo-isomerization of Anabaena sensory rhodopsin.

Anabaena sensory rhodopsin (ASR) is a particular microbial retinal protein for which light-adaptation leads to the ability to bind both the all-trans, 15-anti (AT) and the 13-cis, 15-syn (13C) isomers of the protonated Schiff base of retinal (PSBR). In the context of obtaining insight into the mechanisms by which retinal proteins catalyse the PSBR photo-isomerization reaction, ASR is a model system allowing to study, within the same protein, the protein-PSBR interactions for two different PSBR conformers at the same time. A detailed analysis of the vibrational spectra of AT and 13C, and their photo-products in wild-type ASR obtained through femtosecond (pump-) four-wave-mixing is reported for the first time, and compared to bacterio- and channelrhodopsin. As part of an extensive study of ASR mutants with blue-shifted absorption spectra, we present here a detailed computational analysis of the origin of the mutation-induced blue-shift of the absorption spectra, and identify electrostatic interactions as dominating steric effects that would entail a red-shift. The excited state lifetimes and isomerization reaction times (IRT) for the three mutants V112N, W76F, and L83Q are studied experimentally by femtosecond broadband transient absorption spectroscopy. Interestingly, in all three mutants, isomerization is accelerated for AT with respect to wild-type ASR, and this the more, the shorter the wavelength of maximum absorption. On the contrary, the 13C photo-reaction is slightly slowed down, leading to an inversion of the ESLs of AT and 13C, with respect to wt-ASR, in the blue-most absorbing mutant L83Q. Possible mechanisms for these mutation effects, and their steric and electrostatic origins are discussed.

The role of the ER stress-response protein PERK in rhodopsin retinitis pigmentosa.

Mutations in rhodopsin, the light-sensitive protein of rod cells, are the most common cause of dominant retinitis pigmentosa (RP), a type of inherited blindness caused by the dysfunction and death of photoreceptor cells. The P23H mutation, the most frequent single cause of RP in the USA, causes rhodopsin misfolding and induction of the unfolded protein response (UPR), an adaptive ER stress response and signalling network that aims to enhance the folding and degradation of misfolded proteins to restore proteostasis. Prolonged UPR activation, and in particular the PERK branch, can reduce protein synthesis and initiate cell death through induction of pro-apoptotic pathways. Here, we investigated the effect of pharmacological PERK inhibition on retinal disease process in the P23H-1 transgenic rat model of retinal degeneration. PERK inhibition with GSK2606414A led to an inhibition of eIF2α phosphorylation, which correlated with reduced ERG function and decreased photoreceptor survival at both high and low doses of PERK inhibitor. Additionally, PERK inhibition increased the incidence of inclusion formation in cultured cells overexpressing P23H rod opsin, and increased rhodopsin aggregation in the P23H-1 rat retina, suggesting enhanced P23H misfolding and aggregation. In contrast, treatment of P23H-1 rats with an inhibitor of eIF2α phosphatase, salubrinal, led to improved photoreceptor survival. Collectively, these data suggest the activation of PERK is part of a protective response to mutant rhodopsin that ultimately limits photoreceptor cell death.

Microbial Rhodopsins: Diversity, Mechanisms, and Optogenetic Applications.

Microbial rhodopsins are a family of photoactive retinylidene proteins widespread throughout the microbial world. They are notable for their diversity of function, using variations of a shared seven-transmembrane helix design and similar photochemical reactions to carry out distinctly different light-driven energy and sensory transduction processes. Their study has contributed to our understanding of how evolution modifies protein scaffolds to create new protein chemistry, and their use as tools to control membrane potential with light is fundamental to optogenetics for research and clinical applications. We review the currently known functions and present more in-depth assessment of three functionally and structurally distinct types discovered over the past two years: (a) anion channelrhodopsins (ACRs) from cryptophyte algae, which enable efficient optogenetic neural suppression; (b) cryptophyte cation channelrhodopsins (CCRs), structurally distinct from the green algae CCRs used extensively for neural activation and from cryptophyte ACRs; and

Conformational states of HAMP domains interacting with sensory rhodopsin membrane systems: an integrated all-atom and coarse-grained molecular dynamics simulation approach.

Understanding the downstream signaling mechanism of sensory rhodopsin and its cognate transducer complex (srII-htrII) has long been a challenge in the field of photoreceptor research. Here, an integration of all-atom and coarse-grained (CG) molecular dynamics (MD) simulations in different srII-htrII complex states is carried out. It is shown that the cytoplasmic four-helix HAMP dimer gives rise to a gear-box model interaction with discrete hydrophobic packing in Natronomonas pharaonis (Np). Structural analysis in all-atom and CG-MD reveals a stable conformational state in the physiological environment (323 K and 1.15 M salt). Comparative analysis in the ground and intermediate state conformations reveals substantial inter-HAMP interactions in the intermediate state with uniform clockwise (+10° to +30°) and counterclockwise (-20° to -40°) rotations in the α1 helix and the α2 helix of the monomer, respectively. Low temperature and low salt environments (283 K and 0.15 M) significantly affect srII-htrII binding affinity in both states with unusual helix bending. The distinguished control cable, knob-into-holes packing and piston-like movements in HAMP helices are found in the intermediate state complex. The N-terminal htrII (159 residues) coupled with srII yields a binding energy (ΔGbind) of -309.22, -436.53 and -331.11 kJ mol-1 in the MM/PBSA calculation for the NphtrII homodimer, the NpsrII-htrII ground state conformation and the NpsrII-htrII intermediate state conformation, respectively. Only the HAMP1 domain shows a very low ΔGbind value (-21.03 kJ mol-1) for the ground state in comparison to that for the intermediate state (-54.68 kJ mol-1). The structural analysis highlights the key residues that include Y199srII, T189srII, E43htrII, T86htrII, M100htrII, E116htrII, E126htrII and S130htrII for complex stabilization and signal transduction.

Protective effects of sustained transscleral unoprostone delivery against retinal degeneration in S334ter rhodopsin mutant rats.

It has been suggested that unoprostone isopropyl (UNO) has potent neuroprotective activity in the retina. The effect of sustained transscleral UNO delivery to the posterior segment of the eye on photoreceptor degeneration was evaluated. UNO was loaded into a device made of poly(ethyleneglycol) dimethacrylate by polydimethylsiloxane mold-based UV-curing. The amount of UNO diffusing from these devices was measured using high-performance liquid chromatography. The polymeric devices that released UNO at 1.8 µg/day were implanted on the sclerae of S334ter rats at postnatal 21 days, and electroretinograms (ERGs) were compared with those of topical application and placebo devices. Retinal thickness was evaluated by histological examination. Western blots of specimens 4 weeks after implantation were performed. ERGs showed that the UNO-loaded device prevented the reduction of ERG amplitudes 2 and 4 weeks after implantation, compared with results using a placebo device or topical application. Histological examination showed that the UNO-loaded device prevented the reduction of retinal thickness, and Western blots of specimens indicated that the UNO-loaded device decreased expression of ERK1/2, phosphorylated ERK1/2, and caspase-3. A device that provided sustained UNO administration protected against retinal degeneration in rhodopsin mutant rats, and thus, may have translational potential as a sustainable method to administer drugs to treat retinitis pigmentosa. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 104B: 1730-1737, 2016.

Structure of an Inward Proton-Transporting Anabaena Sensory Rhodopsin Mutant: Mechanistic Insights.

Microbial rhodopsins are light-activated, seven-α-helical, retinylidene transmembrane proteins that have been identified in thousands of organisms across archaea, bacteria, fungi, and algae. Although they share a high degree of sequence identity and thus similarity in structure, many unique functions have been discovered and characterized among them. Some function as outward proton pumps, some as inward chloride pumps, whereas others function as light sensors or ion channels. Unique among the microbial rhodopsins characterized thus far, Anabaena sensory rhodopsin (ASR) is a photochromic sensor that interacts with a soluble 14-kDa cytoplasmic transducer that is encoded on the same operon. The sensor itself stably interconverts between all-trans-15-anti and 13-cis-15-syn retinal forms depending on the wavelength of illumination, although only the former participates in a photocycle with a signaling M intermediate. A mutation in the cytoplasmic half-channel of the protein, replacing Asp217 with Glu (D217E), results in the creation of a light-driven, single-photon, inward proton transporter. We present the 2.3 Å structure of dark-adapted D217E ASR, which reveals significant changes in the water network surrounding Glu217, as well as a shift in the carbon backbone near retinal-binding Lys210, illustrating a possible pathway leading to the protonation of Glu217 in the cytoplasmic half-channel, located 15 Å from the Schiff base. Crystallographic evidence for the protonation of nearby Glu36 is also discussed, which was described previously by Fourier transform infrared spectroscopy analysis. Finally, two histidine residues near the extracellular surface and their possible role in proton uptake are discussed.

Structurally Distinct Cation Channelrhodopsins from Cryptophyte Algae.

Microbial rhodopsins are remarkable for the diversity of their functional mechanisms based on the same protein scaffold. A class of rhodopsins from cryptophyte algae show close sequence homology with haloarchaeal rhodopsin proton pumps rather than with previously known channelrhodopsins from chlorophyte (green) algae. In particular, both aspartate residues that occupy the positions of the chromophore Schiff base proton acceptor and donor, a hallmark of rhodopsin proton pumps, are conserved in these cryptophyte proteins. We expressed the corresponding polynucleotides in human embryonic kidney (HEK293) cells and studied electrogenic properties of the encoded proteins with whole-cell patch-clamp recording. Despite their lack of residues characteristic of the chlorophyte cation channels, these proteins are cation-conducting channelrhodopsins that carry out light-gated passive transport of Na(+) and H(+). These findings show that channel function in rhodopsins has evolved via multiple routes.

Transient Conformational Changes of Sensory Rhodopsin II Investigated by Vibrational Stark Effect Probes.

Sensory rhodopsin II (SRII) is the primary light sensor in the photophobic reaction of the halobacterium Natronomonas pharaonis. Photoactivation of SRII results in a movement of helices F and G of this seven-helical transmembrane protein. This conformational change is conveyed to the transducer protein (HtrII). Global changes in the protein backbone have been monitored by IR difference spectroscopy by recording frequency shifts in the amide bands. Here we investigate local structural changes by judiciously inserting thiocyanides at different locations of SRII. These vibrational Stark probes absorb in a frequency range devoid of any protein vibrations and respond to local changes in the dielectric, electrostatics, and hydrogen bonding. As a proof of principle, we demonstrate the use of Stark probes to test the conformational changes occurring in SRII 12 ms after photoexcitation and later. Thus, a methodology is provided to trace local conformational changes in membrane proteins by a minimal invasive probe at the high temporal resolution inherent to IR spectroscopy.

Cysteine-Specific Labeling of Proteins with a Nitroxide Biradical for Dynamic Nuclear Polarization NMR.

Dynamic nuclear polarization (DNP) enhances the signal in solid-state NMR of proteins by transferring polarization from electronic spins to the nuclear spins of interest. Typically, both the protein and an exogenous source of electronic spins, such as a biradical, are either codissolved or suspended and then frozen in a glycerol/water glassy matrix to achieve a homogeneous distribution. While the use of such a matrix protects the protein upon freezing, it also reduces the available sample volume (by ca. a factor of 4 in our experiments) and causes proportional NMR signal loss. Here we demonstrate an alternative approach that does not rely on dispersing the DNP agent in a glassy matrix. We synthesize a new biradical, ToSMTSL, which is based on the known DNP agent TOTAPOL, but also contains a thiol-specific methanethiosulfonate group to allow for incorporating this biradical into a protein in a site-directed manner. ToSMTSL was characterized by EPR and tested for DNP of a heptahelical transmembrane protein, Anabaena sensory rhodopsin (ASR), by covalent modification of solvent-exposed cysteine residues in two (15)N-labeled ASR mutants. DNP enhancements were measured at 400 MHz/263 GHz NMR/EPR frequencies for a series of samples prepared in deuterated and protonated buffers and with varied biradical/protein ratios. While the maximum DNP enhancement of 15 obtained in these samples is comparable to that observed for an ASR sample cosuspended with ~17 mM TOTAPOL in a glycerol-d8/D2O/H2O matrix, the achievable sensitivity would be 4-fold greater due to the gain in the filling factor. We anticipate that the DNP enhancements could be further improved by optimizing the biradical structure. The use of covalently attached biradicals would broaden the applicability of DNP NMR to structural studies of proteins.

Sexual dimorphism and natural variation within and among species in the Drosophila retinal mosaic.

BACKGROUND: Insect compound eyes are composed of ommatidia, which contain photoreceptor cells that are sensitive to different wavelengths of light defined by the specific rhodopsin proteins that they express. The fruit fly Drosophila melanogaster has several different ommatidium types that can be localised to specific retinal regions, such as the dorsal rim area (DRA), or distributed stochastically in a mosaic across the retina, like the 'pale' and 'yellow' types. Variation in these ommatidia patterns very likely has important implications for the vision of insects and could underlie behavioural and environmental adaptations. However, despite the detailed understanding of ommatidia specification in D. melanogaster, the extent to which the frequency and distribution of the different ommatidium types vary between sexes, strains and species of Drosophila is not known. RESULTS: We investigated the frequency and distribution of ommatidium types based on rhodopsin protein expression, and the expression levels of rhodopsin transcripts in the eyes of both sexes of different strains of D. melanogaster, D. simulans and D. mauritiana. We found that while the number of DRA ommatidia was invariant, Rh3 expressing ommatidia were more frequent in the larger eyes of females compared to the males of all species analysed. The frequency and distribution of ommatidium types also differed between strains and species. The D. simulans strain ZOM4 has the highest frequency of Rh3 expressing ommatidia, which is associated with a non-stochastic patch of pale and odd-coupled ommatidia in the dorsal-posterior of their eyes. CONCLUSIONS: Our results show that there is striking variation in the frequency and distribution of ommatidium types between sexes, strains and species of Drosophila. This suggests that evolutionary changes in the underlying regulatory mechanisms can alter the distribution of ommatidium types to promote or restrict their expression in specific regions of the eye within and between species, and that this could cause differences in vision among these flies.

His166 is the Schiff base proton acceptor in attractant phototaxis receptor sensory rhodopsin I.

Photoactivation of attractant phototaxis receptor sensory rhodopsin I (SRI) in Halobacterium salinarum entails transfer of a proton from the retinylidene chromophore's Schiff base (SB) to an unidentified acceptor residue on the cytoplasmic half-channel, in sharp contrast to other microbial rhodopsins, including the closely related repellent phototaxis receptor SRII and the outward proton pump bacteriorhodopsin, in which the SB proton acceptor is an aspartate residue salt-bridged to the SB in the extracellular (EC) half-channel. His166 on the cytoplasmic side of the SB in SRI has been implicated in the SB proton transfer reaction by mutation studies, and mutants of His166 result in an inverted SB proton release to the EC as well as inversion of the protein's normally attractant phototaxis signal to repellent. Here we found by difference Fourier transform infrared spectroscopy the appearance of Fermi-resonant X-H stretch modes in light-minus-dark difference spectra; their assignment with (15)N labeling and site-directed mutagenesis demonstrates that His166 is the SB proton acceptor during the photochemical reaction cycle of the wild-type SRI-HtrI complex.