Pharaonis phoborhodopsin binds to its cognate truncated transducer even in the presence of a detergent with a 1:1 stoichiometry

Pharaonis phoborhodopsin (ppR) (also pharaonis sensory rhodopsin II) is a receptor of the negative phototaxis of Natronobacterium pharaonis. ppR forms a complex with its pharaonis halobacterial transducer (pHtrII), and this complex transmits the light signal to the sensory system in the cytoplasm. The expressed C-terminal-His tagged ppR and C-terminal-His tagged truncated pHtrII (t-Htr) in Escherichia coli (His means the 6x histidine tag) form a complex even in the presence of 0.1% of n-dodecyl-beta-D-maltoside, and the M-decay of the complex became about twice slower than that of ppR alone. The photocycling rates under varying concentration ratios of ppR to t-Htr in the presence of detergent were measured. The data were analyzed on the following assumptions: (1) the M-decay of both ppR alone and the complex followed a single exponential decay with different time constants; and (2) the M-decay under varying concentration ratios of ppR to t-Htr, therefore, followed a biexponential decay function which combined the decay of the free ppR and that of the complex as photoreactive species. From these analyses we estimated the dissociation constant (15.2 +/- 1.8 microM) and the number of binding sites (1.2 +/- 0.08).     

Tryptophan 171 in Pharaonis phoborhodopsin (sensory rhodopsin II) interacts with the chromophore retinal and its substitution with alanine or threonine slowed down the decay of M- and O-intermediate

Pharaonis phoborhodopsin (ppR), also called pharaonis sensory rhodopsin II, NpSRII, is a photoreceptor for the photophobic response of Natronomonas pharaonis. Tryptophan 182 (W182) of bacteriorhodopsin (bR) is near the chromophore retinal and has been suggested to interact with retinal during the photoreaction and also to be involved in the hydrogen-bonding network around the retinal. W182 of bR is conserved in ppR as tryptophan 171 (W171). To elucidate whether W171 of ppR interacts with retinal during the photoreaction and/or is involved in the hydrogen-bonding network as in bR, we formed W171-substituted mutants of ppR, W171A and W171T. Our low-temperature spectroscopic study has revealed that the substitution of W171 to Ala or Thr resulted in the stabilization of M- and O-intermediates. The stability of M and absorption spectral changes during the M-decay were different depending on the substituted residue. These findings suggest that W171 in ppR interacts with retinal and the degree of the interaction depends on the substituted residues, which might be rate determining in the M-decay. In addition, the involvement of W171 in the hydrogen-bonding network is suggested by the O-decay. We also found that glycerol slowed the decay of M and not of O.     

Arg-72 of pharaonis phoborhodopsin (sensory rhodopsin II) is important for the maintenance of the protein structure in the solubilized states

In bacteriorhodopsin (bR), Arg-82bR has been proven to be a very important residue for functional role of this light-driven proton pump. The arginine residue at this position is a super-conserved residue among archaeal rhodopsins. pharaonis phoborhodopsin (ppR; or called as "pharaonis sensory rhodopsin II") has its absorption maximum at 498 nm and acts as a sensor in the membrane of Natronobacterium pharaonis, mediating the negative phototaxis from the light of wavelength shorter than 520 nm. To investigate the role of the arginine residue (Arg-72ppR) of ppR corresponding to Arg-82bR, mutants whose Arg-72ppR was replaced by alanine (R72A), lysine (R72K), glutamine (R72Q) and serine (R72S) were prepared. These mutants were unstable in low concentrations of NaCl and lost their color gradually when the proteins were solubilized with 0.1% n-dodecyl-beta-D-maltoside. The order of instability was R72S > R72A > R72K > R72Q > the wild type. The rates of denaturation were reduced in a solution of high concentrations of monovalent anions.     

Interaction of Natronobacterium pharaonis phoborhodopsin (sensory rhodopsin II) with its cognate transducer probed by increase in the thermal stability

Pharaonis phoborhodopsin (ppR, also called Natronobacterium pharaonis sensory rhodopsin II) and its transducer protein, pharaonis halobacterial transducer of ppR (pHtrII), form a signaling complex, and light signals are transmitted from the sensor to the transducer by the protein-protein interaction. A truncated pHtrII(1-159) consisting of intramembrane helices (expressing amino acid residues from the first to the 159th position) and ppR form the complex in a solution containing 0.1% n-dodecyl-beta-D-maltoside. At 75-85 degrees C, the time-dependent color loss of ppR was caused by denaturation. We found that pHtrII(1-159) retarded the denaturation rate of ppR. This increase in the thermal stability was used as a probe for the binding ability in the dark. Tyr199 of ppR and Asn74 of pHtrII(1-114) were proposed as amino acid residues interacting with each other through hydrogen bonding. Then,ppR and pHtrII(1-159) mutants at these positions were prepared to examine the effect on the binding in the dark. The wild-type and Y199F mutant can bind pHtrII(1-159), suggesting that the hydrogen bonding between these specific amino acid residues may not be the only cause of the binding, but the hydrophobic interaction via phenyl ring of ppR may contribute dominantly.     

The lifetimes of Pharaonis phoborhodopsin signaling states depend on the rates of proton transfers--effects of hydrostatic pressure and stopped flow experiments

Pharaonis phoborhodopsin (ppR), a negative phototaxis receptor of Natronomonas pharaonis, undergoes photocycle similar to the light-driven proton pump bacteriorhodopsin (BR), but the turnover rate is much slower due to much longer lifetimes of the M and O intermediates. The M decay was shown to become as fast as it is in BR in the L40T/F86D mutant. We examined the effects of hydrostatic pressure on the decay of these intermediates. For BR, pressure decelerated M decay but slightly affected O decay. In contrast, with ppR and with its L40T/F86D mutant, pressure slightly affected M decay but accelerated O decay. Clearly, the pressure-dependent factors for M and O decay are different in BR and ppR. In order to examine the deprotonation of Asp75 in unphotolyzed ppR we performed stopped flow experiments. The pH jump-induced deprotonation of Asp75 occurred with 60 ms, which is at least 20 times slower than deprotonation of the equivalent Asp85 in BR and about 10-fold faster than the O decay of ppR. These data suggest that proton transfer is slowed not only in the cytoplasmic channel but also in the extracellular channel of ppR and that the light-induced structural changes in the O intermediate of ppR additionally decrease this rate.     

Effects of three characteristic amino acid residues of pharaonis phoborhodopsin on the absorption maximum

Phoborhodopsin (pR or sensory rhodopsin II, sRII) or pharaonis phoborhodopsin (ppR or pharaonis sensory rhodopsin II, psRII) has a unique absorption maximum (lambda max) compared with three other archaeal rhodopsins: lambda max of pR or ppR at ca 500 nm and others at 560-590 nm. Alignment of amino acid sequences revealed three sites characteristic of the shorter wavelength-absorbing pigments. The amino acids of these three sites are conserved completely among archaeal rhodopsins having longer lambda max, and are different from those of pR or ppR. We replaced these amino acids of ppR with amino acids corresponding to those of bacteriorhodopsin, Val-108 to Met, Gly-130 to Ser and Thr-204 to Ala. The lambda max of V108M mutant was 502 nm with a slight redshift. G130S and T204A mutants had lambda max of 503 and 508 nm, respectively. Thus, each site contributes only a small effect to the color tuning. We then constructed three double mutants and one triple mutant. The opsin-shifts of these mutants suggest that Val-108 and Thr-204 or Gly-130 are synergistic, and that Gly-130 and Thr-204 work additively. Even in the triple mutant, the lambda max was 515 nm, an opsin-shift only ca 30% of the shift value from 500 to 560 nm. This means that there is another yet unidentified factor responsible for the color tuning.     

Association between a photo-intermediate of a M-lacking mutant D75N of pharaonis phoborhodopsin and its cognate transducer

Pharaonis phoborhodopsin (ppR or pharaonis sensory rhodopsin II) is a receptor of the negative phototaxis of Natronobacterium pharaonis and forms a complex with its transducer pHtrII in membranes. Flash-photolyis of a D75N mutant did not yield the M-intermediate, but an O-like intermediate is observed in a ms time range. We examined the interaction between the D75N of ppR and t-Htr (truncated pHtrII). These formed a complex in the presence of 0.1% n-dodecyl-beta-maltoside, and the association accelerated the decay of the O of D75N from 15 to 56 s(-1). From the decay time constants under varying ratios of D75N and t-Htr, n, the molar ratio of D75N/t-Htr in the complex, and K(D), the dissociation constant, were estimated. The value of n was unity and K(D) was estimated to 146 nM. This K(D) value can be considered to be the association between the photo-intermediate and t-Htr, which is deduced by the method of estimation. Previously we (Photochem. Photobiol. 74 (2001) 489) reported a K(D) of 15 microM for the interaction between the wild-type and t-Htr by means of the change in M-decay rates. Therefore, this value should be the K(D) value for the interaction between M of the wild-type and t-Htr.     

Protein-protein interaction of a Pharaonis halorhodopsin mutant forming a complex with Pharaonis halobacterial transducer protein II detected by Fourier-transform infrared spectroscopy

Pharaonis halorhodopsin (pHR) functions as a light-driven inward chloride ion pump in Natoronomonas pharaonis, while pharaonis phoborhodopsin (ppR; also called pharaonis sensory rhodopsin II, pSRII), is a light sensor for negative phototaxis. ppR forms a 2:2 complex with its cognate transducer protein (pHtrII) through intramembranous hydrogen bonds: Tyr199(ppR)-Asn74(pHtrII) and Thr189(ppR)-Glu43 (pHtrII), Ser62(pHtrII). It was reported that a pHR mutant (P240T/F250Y), which possesses the hydrogen-bonding sites, impairs its pumping activity upon complexation with pHtrII. In this study, effect of the complexation with pHtrII on the structural changes upon formation of the K, L(1) and L(2) intermediates of pHR was investigated by use of Fourier-transform infrared spectroscopy. The vibrational changes of Tyr250(pHR) and Asn74(pHtrII) were detected for the L(1) and L(2) intermediates, supporting that Tyr250(pHR) forms a hydrogen bond with Asn74(pHtrII) as similarly to Tyr199(ppR). The conformational changes of the retinal chromophore were never affected by complexation with pHtrII, but amide-I vibrations were clearly different in the absence and presence of pHtrII. The molecular environment around Asp156(pHR) in helix D is also slightly affected. These additional structural changes are probably related to blocking of translocation of a chloride ion from the extracellular to the cytoplasmic side during the photocycle.     

Application of fluorescence resonance energy transfer (FRET) to investigation of light-induced conformational changes of the phoborhodopsin/transducer complex

The photoreceptor phoborhodopsin (ppR; also called sensory rhodopsin II) forms a complex with its cognate the Halobacterial transducer II (pHtrII) in the membrane, through which changes in the environmental light conditions are transmitted to the cytoplasm in Natronomonas pharaonis to evoke negative phototaxis. We have applied a fluorescence resonance energy transfer (FRET)-based method for investigation of the light-induced conformational changes of the ppR/pHtrII complex. Several far-red dyes were examined as possible fluorescence donors or acceptors because of the absence of the spectral overlap of these dyes with all the photointermediates of ppR. The flash-induced changes of distances between the donor and an acceptor linked to cysteine residues which were genetically introduced at given positions in pHtrII(1-159) and ppR were determined from FRET efficiency changes. The dye-labeled complex was studied as solubilized in 0.1% n-dodecyl-beta-D-maltoside (DDM). The FRET-derived changes in distances from V78 and A79 in pHtrII to V185 in ppR were consistent with the crystal structure data (Moukhametzianov, R. et al. [2006] Nature, 440, 115-119). The distance from D102 in pHtrII linker region to V185 in ppR increased by 0.33 angstroms upon the flash excitation. These changes arose within 70 ms (the dead time of instrument) and decayed with a rate of 1.1 +/- 0.2 s. Thus, sub-angstrom-scale distance changes in the ppR/pHtrII complex were detected with this FRET-based method using far-red fluorescent dyes; this method should be a valuable tool to investigate conformation changes in the transducer, in particular its dynamics.     

Dynamics change of phoborhodopsin and transducer by activation: study using D75N mutant of the receptor by site-directed solid-state 13C NMR

Pharaonis phoborhodopsin (ppR or sensory rhodopsin II) is a negative phototaxis receptor of Natronomonas pharaonis, and forms a complex, which transmits the photosignal into cytoplasm, with its cognate transducer (pHtrII). We examined a possible local dynamics change of ppR and its D75N mutant complexed with pHtrII, using solid-state (13)C NMR of [3-(13)C]Ala- and [1-(13)C]Val-labeled preparations. We distinguished Ala C(beta) (13)C signals of relatively static stem (Ala221) in the C-terminus of the receptors from those of flexible tip (Ala228, 234, 236 and 238), utilizing a mutant with truncated C-terminus. The local fluctuation frequency at the C-terminal tip was appreciably decreased when ppR was bound to pHtrII, while it was increased when D75N, that mimics the signaling state because of disrupted salt bridge between C and G helices prerequisite for the signal transfer, was bound to pHtrII. This signal change may be considered with the larger dissociation constant of the complex between pHtrII and M-state of ppR. At the same time, it turned out that fluctuation frequency of cytoplasmic portion of pHtrII is lowered when ppR is replaced by D75N in the complex with pHtrII. This means that the C-terminal tip partly participates in binding with the linker region of pHtrII in the dark, but this portion might be released at the signaling state leading to mutual association of the two transducers in the cytoplasmic regions within the ppR/pHtrII complex.     

Effect of polarization on the opsin shift in rhodopsins. 1. A combined QM/QM/MM model for bacteriorhodopsin and pharaonis sensory rhodopsin II

The optical and IR-spectroscopic properties of the protonated Schiff base of retinal are highly sensitive to the electrostatic environment. This feature makes retinal a useful probe to study structural differences and changes in rhodopsins. It also raises an interest to theoretically predict the spectroscopic response to mutation and structural evolution. Computational models appropriate for this purpose usually combine sophisticated quantum mechanical (QM) methods with molecular mechanics (MM) force fields. In an effort to test and improve the accuracy of these QM/MM models, we consider in this article the effects of polarization and inter-residual charge transfer within the binding pocket of bacteriorhodopsin (bR) and pharaonis sensory rhodopsin II (psRII, also called pharaonis phoborhodopsin, ppR) on the excitation energy using an ab initio QM/QM/MM approach. The results will serve as reference for assessing empirical polarization models in a consecutive article.     

NANOSECOND LASER PHOTOLYSIS OF PHOBORHODOPSIN: FROM Natronobacterium pharaonis APPEARANCE OF KL AND L INTERMEDIATES IN THE PHOTOCYCLE AT ROOM TEMPERATURE

Abstract— Photochemical and subsequent thermal reactions of pharaonis phoborhodopsin (ppR; absorption maximum, 498 nm) from Natronobacrerium pharaonis were investigated by nanosecond laser photolysis at 20°C. The experimental results clearly showed the presence of two intermediates in the photocycle of ppR besides the K, M and O intermediates detected previously. One was formed immediately after the excitation of ppR with a blue pulse (pulse width, 17 ns; wavelength, 460 nm), and the other was formed by the thermal reaction of this species. The new intermediates' absorption maxima were 512 and 488 nm, their extinction coefficients were 0.85- and 0.68-times smaller than that of ppR, and their lifetimes were 990 ns and 32 μs, respectively. The absorption and kinetic characteristics of these intermediates relative to ppR were similar to those of the KL and L intermediates of bacteriorhodopsin (bR). The formation of KL intermediates from both ppR and bR were observed only at room temperatures. On the other hand, the formation of L intermediate of bR was observed at both of room and low temperature, whereas that from ppR only at room temperature. The unique formation of L intermediate of ppR at room temperature is discussed in relation to high thermal stability of K intermediate of ppR.     

SHAPE OF THE CHROMOPHOIW BINDING SITE IN pharaonis PHOBORHODOPSIN FROM A STUDY USING RETINAL ANALOGS

Abstract To investigate the shape of the chromophore binding site of pharaonis phoborhodopsin (ppR), ppR-opsin was incubated with five ring-modified retinal analogs: an acyclic retinal, phenylretinal, α-retinal, cyclohexylretinal and 5-isopropyl-α-retinal. The experimental results were compared with those obtained from bacteriorhodopsin-opsin (bR-opsin) and the same retinal analogs. It was suggested that ring chain conformation is important in affecting the spectral shoulder unique for the absorption spectrum of ppR. The rate of pigment formation depended greatly on the analogs used with the planar analogs showing rapid formation. Thus, we concluded that the space of the retinal binding site of ppR is restricted to the plane of the cyclohexenyl ring of the chromophore, whereas that of bR is less restricted.     

Participation of the surface structure of Pharaonis phoborhodopsin, ppR and its A149S and A149V mutants, consisting of the C-terminal alpha-helix and E-F loop, in the complex-formation with the cognate transducer pHtrII, as revealed by site-directed 13C solid-state NMR

We have recorded 13C solid state NMR spectra of [3-13C]Ala-labeled pharaonis phoborhodopsin (ppR) and its mutants, A149S and A149V, complexed with the cognate transducer pharaonis halobacterial transducer II protein (pHtrII) (1-159), to gain insight into a possible role of their cytoplasmic surface structure including the C-terminal alpha-helix and E-F loop for stabilization of the 2:2 complex, by both cross-polarization magic angle spinning (CP-MAS) and dipolar decoupled (DD)-MAS NMR techniques. We found that 13C CP-MAS NMR spectra of [3-13C]Ala-ppR, A149S and A149V complexed with the transducer pHtrII are very similar, reflecting their conformation and dynamics changes caused by mutual interactions through the transmembrane alpha-helical surfaces. In contrast, their DD-MAS NMR spectral features are quite different between [3-13C]Ala-A149S and A149V in the complexes with pHtrII: 13C DD-MAS NMR spectrum of [3-13C]Ala-A149S complex is rather similar to that of the uncomplexed form, while the corresponding spectral feature of A149V complex is similar to that of ppR complex in the C-terminal tip region. This is because more flexible surface structure detected by the DD-MAS NMR spectra are more directly influenced by the dynamics changes than the CP-MAS NMR. It turned out, therefore, that an altered surface structure of A149S resulted in destabilized complex as viewed from the 13C NMR spectrum of the surface areas, probably because of modified conformation at the corner of the helix E in addition to the change of hydropathy. It is, therefore, concluded that the surface structure of ppR including the C-terminal alpha-helix and the E-F loops is directly involved in the stabilization of the complex through conformational stability of the helix E.     

Steric constraint in the primary photoproduct of an archaeal rhodopsin from regiospecific perturbation of C-D stretching vibration of the retinyl chromophore

In visual and archaeal rhodopsins, light energy is stored in the chromophore-protein interaction after retinal photoisomerization. This paper reports a novel method to monitor the steric constraint after retinal isomerization by use of enhanced C-D stretching vibrations. In the difference FTIR spectra between an archaeal light-sensor pharaonis phoborhodopsin (ppR) and the primary K intermediate at 77 K, no peaks were observed in the 2160-2330 cm-1 region for deuterated retinals at position 7, 8, 10, 11, 12, and 15, whereas a strong peak appeared at 2244 cm-1 for the K intermediate of ppR possessing a C14-D-labeled retinal. The 2244-cm-1 band is assigned as the C14-D stretching vibration, and enhanced absorption in the K state probably originates from the local steric constraint at the C14-D position (also possible electrostatic field effects) after the C13=C14 double bond rotation.     

Interaction of the halobacterial transducer to a halorhodopsin mutant engineered so as to bind the transducer: Cl- circulation within the extracellular channel

An alkali-halophilic archaeum, Natronomonas pharaonis, contains two rhodopsins that are halorhodopsin (phR), a light-driven inward Cl- pump and phoborhodopsin (ppR), the receptor of negative phototaxis functioning by forming a signaling complex with a transducer, pHtrII (Sudo Y. et al., J. Mol. Biol. 357 [2006] 1274). Previously, we reported that the phR double mutant, P240T/F250Y(phR), can bind with pHtrII. This mutant itself can transport Cl-, while the net transport was stopped upon formation of the complex. The flash-photolysis data were analyzed by a scheme in which phR --> 4 P1 --> P2 --> 4 P3 --> P4 --> phR. The P3 of the wild-type and the double mutant contained two components, X- and O-intermediates. After the complex formation, however, the P3 of the double mutant lacked the X-intermediate. These observations imply that the X-intermediate (probably the N-intermediate) is the state having Cl- in the cytoplasmic binding site and that the complex undergoes an extracellular Cl- circulation because of the inhibition of formation of the X-intermediate.     

The distinct signaling mechanisms of microbial sensory rhodopsins in Archaea, Eubacteria and Eukarya

Most of the known archaeal-type microbial rhodopsins are retinal-binding ion transporters, such as bacteriorhodopsin (BR) and proteorhodopsin (PR). Their identification is the result of extensive studies of their photochemical and biophysical properties. The cells containing these pigments, however, use other microbial rhodopsins as photosensors to monitor environmental light signals. From the early studies of sensory rhodopsin I (HsSRI) in Halobacterium salinarum and sensory rhodopsin II (NpSRII) in Natronomonas pharaonis, we now know that several microbial sensory rhodopsins in the other major domain of life relay information on light intensity and quality to the cell. Three of the most studied photosensory transduction mechanisms of these microbial rhodopsins are dealt with in this review. We discuss recent progress in the understanding of genomic organization, photochemical properties and photosignaling mechanisms with respect to biological function.     

NANOSECOND LASER PHOTOLYSIS OF RHODOPSIN AND ISORHODOPSIN

Kinetic and spectral measurements have been carried out on the primary intermediate in the photolysis of rhodopsin and isorhodopsin, initiated by a 457 nm, 6 ns (FWHM) laser pulse. In rhodopsin the kinetic decay of bathorhodopsin was found to be 140 ± 15 ns at 20°C. The decay of bathorhodopsin to lumirhodopsin has an activation energy of 51 ± 4 kJ/mol (12.2 ± 1 kcal/mol). The decay kinetics of bathorhodopsin were found to be the same for rhodopsin in membrane and detergent solubilized suspensions. The kinetic decay of the batho product in the photolysis of isorhodopsin was found to be the same as rhodopsin.
The corrected transient spectrum 50 ns following excitation in rhodopsin has two peaks near 560 and 440 nm. A peak was also observed in isorhodopsin near 550 nm at 50 ns following excitation but no transient was observed in the blue. The 550 nm peak in isorhodopsin has an intensity similar to that in rhodopsin indicating that the quantum yields for the formation of batho products of rhodopsin and isorhodopsin are similar under the irradiation conditions used here. Transient spectra for rhodopsin and isorhodopsin 1 μs following excitation are also different. In isorhodopsin the corrected transient spectrum has a peak at 500 nm, similar to low temperature steady state irradiation spectra. The 1 μs transient spectrum in rhodopsin is more intense than in isorhodopsin and shows a peak at 475 nm.     

Vibrational spectroscopy and dynamics of azide ion in ionic liquid and dimethyl sulfoxide water mixtures

Steady-state and time-resolved infrared spectroscopy of the azide (N(3)-) anion has been used to characterize aqueous mixtures both with the ionic liquid (IL) 1-butyl-3-methylimidazolium tetrafluoroborate ([BMIM][BF(4)]) and with dimethyl sulfoxide (DMSO). In the DMSO-water mixtures, two anion vibrational bands are observed for low water mole fractions (0 > X(w) > 0.25), which indicates a heterogeneous ion solvation environment. The band at 2000 cm(-1) observed for neat DMSO does not shift but decreases in amplitude as the amount of water is increased. Another band appears at slightly higher frequency at low X(w) (=0.05). As the amount of water is increased, this band shifts to higher frequency and becomes stronger and is attributed to azide with an increasing degree of hydration. At intermediate and high X(w), a single band is observed that shifts almost linearly with water mole fraction toward the bulk water value. The heterogeneity is evident from the infrared pump-probe studies in which the decay times depend on probe frequency at low mole fraction. For the azide spectra in IL-water mixtures, a single azide band is observed for each mole fraction mixture. The azide band shifts almost linearly with mole fraction, indicating nearly ideal mixing behavior. As with the DMSO-water mixtures, the time-resolved IR decay times are probe-frequency-dependent at low mole fraction, again indicating heterogeneous solvation. In both the DMSO and IL mixtures with water, the relaxation times are slower than would be expected from ideal mixing, suggesting that vibrational relaxation of azide is more sensitive than its vibrational frequency to the solvent structure. The results are discussed in terms of preferential solvation and the degree to which the azide shift and vibrational relaxation depend on the degree of water association in the mixtures.     

Photochemistry of a putative new class of sensory rhodopsin (SRIII) coded by xop2 of Haloarcular marismortui

Baliga et al. (2004) [1] reported the existence of a functionally unpredictable opsin gene, named xop2, in Haloarcula marismortui, a holophilic archaeon. Ihara et al. [38] performed molecular phylogenetic analysis and determined that the product of xop2 belonged to a new class of opsins in the sensory rhodopsins. This microbial rhodopsin was therefore named H. marismortui sensory rhodopsin III (HmSRIII). Here, we functionally expressed HmSRIII in Escherichia coli cell membranes to examine the photochemistry. The wavelength of maximum absorption (λ(max)) for HmSRIII was 506nm. We observed a very slow photocycle that completed in ～50s. Intermediates were defined as M (λ(max)～380nm), N (λ(max)～460nm) and O (λ(max)～530nm) 0.01s after the flash excitation. The nomenclature for these intermediates was based on their locations along the absorption maxima of bacteriorhodopsin. Analysis of laser-flash-photolysis data in the presence and absence of azide gave the following results: (1) an equilibrium between N and O was attained, (2) the direct product of the M-decay was O but not N, and (3) the last photo-intermediate (HmSRIII') had a λ(max) similar to that of the original, and its decay rate was very slow. Resonance Raman spectroscopy revealed that this N-intermediate had 13-cis retinal conformation. Proton uptake occurred during the course of M-decay, whereas proton release occurred during the course of O-decay (or exactly N-O equilibrium). Very weak proton-pumping activity was observed whose direction is the same as that of bacteriorhodopsin, a typical light-driven proton pump.