FTIR spectroscopy of the O photointermediate in pharaonis phoborhodopsin

pharaonis phoborhodopsin (ppR; also called pharaonis sensory rhodopsin II, psR-II) is a photoreceptor protein for negative phototaxis in Natronobacterium pharaonis. During the photocycle of ppR, the retinal chromophore is thermally isomerized from the 13-cis to all-trans form. We employed FTIR spectroscopy of ppR at 260 K and pH 5 to reveal that this isomerization occurs upon formation of the O intermediate (ppR(O)) by using ppR samples reconstituted with 12,14-D(2)-labeled retinal. In ppR(O), C=O stretching vibrations of protonated carboxylates newly appear at 1757 (+)/1722 (-) cm(-1) in H(2)O and at 1747 (+)/1718 (-) cm(-1) in D(2)O in addition to the 1765 (+) cm(-1) band of Asp75. Amide I vibrations are basically similar between ppR(M) and ppR(O), whereas unique bands of ppR(O) are also observed such as the negative 1656 cm(-1) band in D(2)O and intense bands at 1686 (-)/1674 (+) cm(-1). In addition, O-D stretching vibrations of water molecules in the entire mid-infrared region are assigned for ppR(M) and ppR(O), the latter being unique for ppR, since it can be detected at low temperature (260 K). The ppR(M) minus ppR difference spectra lack the lowest frequency water band (2215 cm(-1)) observed in the ppR(K) minus ppR spectra, which is probably associated with water that interacts with the negative charges in the Schiff base region. It is likely that the proton transfer from the Schiff base to Asp75 in ppR(M) can be explained by a hydration switch of a water from Asp75 to Asp201, as was proposed for the light-driven proton-pump bacteriorhodopsin (hydration switch model) [Tanimoto, T., Furutani, Y., and Kandori, H. (2003) Biochemistry 42, 2300-2306]. In the transition from ppR(M) to ppR(O), a hydrogen-bonding alteration takes place for another water molecule that forms a strong hydrogen bond.     

Steric constraint in the primary photoproduct of sensory rhodopsin II is a prerequisite for light-signal transfer to HtrII

Sensory rhodopsin II (SRII, also called pharaonis phoborhodopsin, ppR) is responsible for negative phototaxis in Natronomonas pharaonis. Photoisomerization of the retinal chromophore from all- trans to 13- cis initiates conformational changes in the protein, leading to activation of the cognate transducer protein (HtrII). We previously observed enhancement of the C 14-D stretching vibration of the retinal chromophore at 2244 cm (-1) upon formation of the K state and interpreted that a steric constraint occurs at the C 14D group in SRII K. Here, we identify the counterpart of the C 14D group as Thr204, because the C 14-D stretching signal disappeared in T204A, T204S, and T204C mutants as well as a C 14-HOOP (hydrogen out-of-plane) vibration at 864 cm (-1). Although the K state of the wild-type bacteriorhodopsin (BR), a light-driven proton pump, possesses neither 2244 nor 864 cm (-1) bands, both signals appeared for the K state of a triple mutant of BR that functions as a light sensor (P200T/V210Y/A215T). We found a positive correlation between these vibrational amplitudes of the C 14 atom at 77 K and the physiological phototaxis response. These observations strongly suggest that the steric constraint between the C 14 group of retinal and Thr204 of the protein is a prerequisite for light-signal transduction by SRII.     

Interaction of Asn105 with the retinal chromophore during photoisomerization of pharaonis phoborhodopsin

pharaonis phoborhodopsin (ppR; also called pharaonis sensory rhodopsin II, psR-II) is a photoreceptor for negative phototaxis in Natronobacterium pharaonis. ppR has a blue-shifted absorption spectrum with a spectral shoulder, which is highly unique for the archaeal rhodopsin family. The primary reaction of ppR is a cis-trans photoisomerization of the retinal chromophore to form the K intermediate, like the well-studied proton pump bacteriorhodopsin (BR). Recent comparative FTIR spectroscopy of the K states in ppR and BR revealed that more extended structural changes take place in ppR than in BR with respect to chromophore distortion and protein structural changes [Kandori, H., Shimono, K., Sudo, Y., Iwamoto, M., Shichida, Y., and Kamo, N. (2001) Biochemistry 40, 9238-9246]. FTIR spectroscopy of the N105D mutant protein reported here assigns the vibrational bands at 1704 and 1700 cm(-1) as C=O stretches of Asn105 in ppR and ppR(K), respectively. A comparative investigation between ppR and BR further reveals that the structure at position 105 in ppR is similar to that of the corresponding position (Asp115) in BR; this observation is supported by the recent X-ray crystallographic structures of ppR [Luecke, H., Schobert, B., Lanyi, J. K., Spudich, E. N., and Spudich, J. L. (2001) Science 293, 1499-1503; Royant, A., Nollert, P., Edman, K., Neutze, R., Landau, E. M., Pebay-Peyroulla, E., and Navarro, J. (2001) Proc. Natl. Acad. Sci. U.S.A. 98, 10131-10136]. Nevertheless, structural changes upon photoisomerization at position 105 in ppR are greater than those at position 115 in BR. As a consequence of a unique chromophore-protein interaction in ppR, extended protein structural changes accompanying retinal photoisomerization occur, and these include Asn105 which is approximately 7 A from the retinal chromophore.     

Structural changes of pharaonis phoborhodopsin upon photoisomerization of the retinal chromophore: infrared spectral comparison with bacteriorhodopsin

Archaeal rhodopsins possess a retinal molecule as their chromophores, and their light energy and light signal conversions are triggered by all-trans to 13-cis isomerization of the retinal chromophore. Relaxation through structural changes of the protein then leads to functional processes, proton pump in bacteriorhodopsin and transducer activation in sensory rhodopsins. In the present paper, low-temperature Fourier transform infrared spectroscopy is applied to phoborhodopsin from Natronobacterium pharaonis (ppR), a photoreceptor for the negative phototaxis of the bacteria, and infrared spectral changes before and after photoisomerization are compared with those of bacteriorhodopsin (BR) at 77 K. Spectral comparison of the C--C stretching vibrations of the retinal chromophore shows that chromophore conformation of the polyene chain is similar between ppR and BR. This fact implies that the unique chromophore-protein interaction in ppR, such as the blue-shifted absorption spectrum with vibrational fine structure, originates from both ends, the beta-ionone ring and the Schiff base regions. In fact, less planer ring structure and stronger hydrogen bond of the Schiff base were suggested for ppR. Similar frequency changes upon photoisomerization are observed for the C==N stretch of the retinal Schiff base and the stretch of the neighboring threonine side chain (Thr79 in ppR and Thr89 in BR), suggesting that photoisomerization in ppR is driven by the motion of the Schiff base like BR. Nevertheless, the structure of the K state after photoisomerization is different between ppR and BR. In BR, chromophore distortion is localized in the Schiff base region, as shown in its hydrogen out-of-plane vibrations. In contrast, more extended structural changes take place in ppR in view of chromophore distortion and protein structural changes. Such structure of the K intermediate of ppR is probably correlated with its high thermal stability. In fact, almost identical infrared spectra are obtained between 77 and 170 K in ppR. Unique chromophore-protein interaction and photoisomerization processes in ppR are discussed on the basis of the present infrared spectral comparison with BR.     

FTIR spectroscopy of the M photointermediate in pharaonis rhoborhodopsin

pharaonis phoborhodopsin (ppR; also called pharaonis sensory rhodopsin II, psR-II) is a photoreceptor for negative phototaxis in Natronobacterium pharaonis. During the photocycle of ppR, the Schiff base of the retinal chromophore is deprotonated upon formation of the M intermediate (ppR(M)). The present FTIR spectroscopy of ppR(M) revealed that the Schiff base proton is transferred to Asp-75, which corresponds to Asp-85 in a light-driven proton-pump bacteriorhodopsin (BR). In addition, the C==O stretching vibrations of Asn-105 were assigned for ppR and ppR(M). The common hydrogen-bonding alterations in Asn-105 of ppR and Asp-115 of BR were found in the process from photoisomerization (K intermediate) to the primary proton transfer (M intermediate). These results implicate similar protein structural changes between ppR and BR. However, BR(M) decays to BR(N) accompanying a proton transfer from Asp-96 to the Schiff base and largely changed protein structure. In the D96N mutant protein of BR that lacks a proton donor to the Schiff base, the N-like protein structure was observed with the deprotonated Schiff base (called M(N)) at alkaline pH. In ppR, such an N-like (M(N)-like) structure was not observed at alkaline pH, suggesting that the protein structure of the M state activates its transducer protein.     

An amino acid residue (S201) in the retinal binding pocket regulates the photoreaction pathway of phoborhodopsin

Phoborhodopsin from Halobacterium salinarum (salinarum phoborhodopsin, spR also called HsSR II) is a photoreceptor for the negative phototaxis of the bacterium. A unique feature of spR is the formation of a shorter wavelength photoproduct, P480, observed at liquid nitrogen temperature beside the K intermediate. Formation of similar photoproduct has not been reported in the other microbial rhodopsins. This photoproduct showed its maximum absorbance wavelength (λ(max)) at 482 nm and can thermally revert back to spR above -160 °C. It was revealed that P480 is a photoproduct of K intermediate by combination of an irradiation and warming experiment. Fourier transform infrared (FTIR) difference spectrum of P480 from spR in C-C stretching vibration region showed similar features with that of K intermediate, suggesting that P480 has a 13-cis-retinal chromophore. The appearance of a broad positive band at 1214 cm(-1) in the P480-spR spectrum suggested that configuration around C9═C10 likely be different between P480 and K intermediate. Vibrational bands in HOOP region (1035 to 900 cm(-1)) suggested that the chromophore distortion in K intermediate was largely relaxed in P480. The amount of P480 formed by the irradiation was greatly decreased by amino acid replacement of S201 with T, suggesting S201 was involved in the formation of P480. According to the crystal structure of pharaonis phoborhodopsin (ppR), a homologue of spR found in Natronomonas pharaonis, S201 should locate near the C14 of retinal chromophore. Thus, the interaction between S201 and C14 might be the main factor affecting formation of P480.     

FTIR spectroscopy of the complex between pharaonis phoborhodopsin and its transducer protein

pharaonis phoborhodopsin (ppR; also called pharaonis sensory rhodopsin II, psRII) is a photoreceptor for negative phototaxis in Natronobacterium pharaonis. ppR activates the cognate transducer protein, pHtrII, upon absorption of light. ppR and pHtrII form a tight 2:2 complex in the unphotolyzed state, and the interaction is somehow altered during the photocycle of ppR. In this paper, we studied the influence of pHtrII on the structural changes occurring upon retinal photoisomerization in ppR by means of low-temperature FTIR spectroscopy. We trapped the K intermediate at 77 K and compared the ppR(K) minus ppR spectra in the absence and presence of pHtrII. There are no differences in the X-D stretching vibrations (2700-1900 cm(-1)) caused by presence of pHtrII. This result indicates that the hydrogen-bonding network in the Schiff base region is not altered by interaction with pHtrII, which is consistent with the same absorption spectrum of ppR with or without pHtrII. In contrast, the ppR(K) minus ppR infrared difference spectra are clearly influenced by the presence of pHtrII in amide-I (1680-1640 cm(-1)) and amide-A (3350-3250 cm(-1)) vibrations. The identical spectra for the complex of the unlabeled ppR and (13)C- or (15)N-labeled pHtrII indicate that the observed structural changes for the peptide backbone originate from ppR only and are altered by retinal photoisomerization. The changes do not come from pHtrII, implying that the light signal is not transmitted to pHtrII in ppR(K). In addition, we observed D(2)O-insensitive bands at 3479 (-)/3369 (+) cm(-1) only in the presence of pHtrII, which presumably originate from an X-H stretch of an amino acid side chain inside the protein.     

A pharaonis phoborhodopsin mutant with the same retinal binding site residues as in bacteriorhodopsin

pharaonis phoborhodopsin (ppR, also called pharaonis sensory rhodopsin II, psR-II) is a photoreceptor for negative phototaxis in Natronobacterium pharaonis. ppR has a blue-shifted absorption maximum (500 nm) relative those of other archaeal rhodopsins such as the proton-pump bacteriorhodopsin (BR; 570 nm). Among the 25 amino acids that are within 5 A of the retinal chromophore, 10 are different in BR and ppR, and they are presumed to be crucial in determining the color of their chromophores. However, the spectral red shift in a multiple mutant of ppR, in which the retinal binding site was made similar to that of BR (BR/ppR), was smaller than 40% (lambda(max) = 524 nm) than expected. In the paper presented here, we report on low-temperature Fourier transform infrared (FTIR) spectroscopy of BR/ppR, and compare the infrared spectral changes before and after photoisomerization with those for ppR and BR. The C[bond]C stretch and hydrogen out-of-plane (HOOP) vibrations of BR/ppR were similar to those of BR, suggesting that the surrounding protein moiety of BR/ppR becomes like BR. However, BR/ppR exhibited a unique IR band regarding the hydrogen bond of the protonated Schiff base. It has been known that ppR has a stronger hydrogen bond for the Schiff base than BR as judged from the frequency difference between their C[double bond]NH and C[double bond]ND stretches. We now find that replacement of the 10 amino acids of BR with ppR (BR/ppR) does not weaken the hydrogen bond of the Schiff base. Rather, the hydrogen bond in BR/ppR is stronger than that in the native ppR. We conclude that the principal factor of the smaller than expected opsin shift in BR/ppR is the strong association of the Schiff base with the surrounding counterion complex.     

Vibrational modes of the protonated Schiff base in pharaonis phoborhodopsin

pharaonis phoborhodopsin (ppR; also called pharaonis sensory rhodopsin II, psR-II) is a photoreceptor for negative phototaxis in Natronobacterium pharaonis. Recent X-ray crystallographic structures showed that ppR and bacteriorhodopsin (BR), a light-driven proton pump, possess similar molecular environments of the retinal Schiff base. Nevertheless, absorption spectra are different by 70 nm between ppR and BR, suggesting the different chromophore-protein interactions involving the Schiff base region. In this article, we identify frequencies of the Schiff base vibrations in the ppR(K) minus ppR difference spectra by means of low-temperature FTIR spectroscopy of [zeta-(15)N]lysine-labeled ppR. The N-D stretch in D(2)O was found at 2140 and 2091 cm(-1) for ppR, which are shifted to a lower frequency by 32-33 cm(-1) compared to those for BR. This observation indicates the stronger hydrogen bond of the Schiff base in ppR than in BR. The N-D stretch of the Schiff base and O-D stretch of water molecules are located at the different frequencies in ppR, while they appear in the same frequency region in BR [Kandori, H., Belenky, M., and Herzfeld, J. (2002) Biochemistry 41, 6026-6031]. These differences could be correlated with the distorted pentagonal cluster structure in ppR. In contrast, the N-D stretch of ppR(K) was found at 2474 cm(-1), which is close in frequency to that of BR(K). The O-D stretch of Thr79 was also assigned at 2512 and 2474 cm(-1) for ppR and ppR(K), respectively. These frequencies are close to those of BR, suggesting the interaction of Thr79 and Asp75 in ppR is similar to that of Thr89 and Asp85 in BR.     

Hydrogen bonding alteration of Thr-204 in the complex between pharaonis phoborhodopsin and its transducer protein

Pharaonis phoborhodopsin (ppR, also called pharaonis sensory rhodopsin II, psRII) is a receptor for negative phototaxis in Natronobacterium pharaonis. It forms a 2:2 complex with its transducer protein, pHtrII, in membranes and transmits light signals through the change in the protein-protein interaction. We previously found that the ppR(K) minus ppR spectrum in D(2)O possesses vibrational bands of ppR at 3479 (-)/3369 (+) cm(-1) only in the presence of pHtrII [Furutani, Y., Sudo, Y., Kamo, N., and Kandori, H. (2003) Biochemistry 42, 4837-4842]. A D/H-unexchangeable X-H group appears to form a stronger hydrogen bond upon retinal photoisomerization in the ppR-pHtrII complex. This article aims to identify the group by use of various mutant proteins. According to the crystal structure, Tyr-199 of ppR forms a hydrogen bond with Asn-74 of pHtrII in the complex. Nevertheless, the 3479 (-)/3369 (+) cm(-1) bands were preserved in the Y199F mutant, excluding the possibility that the bands are O-H stretches of Tyr-199. On the other hand, Thr-204 and Tyr-174 form a hydrogen bond between the retinal chromophore pocket and the binding surface of the ppR-pHtrII complex. These FTIR measurements revealed that the bands at 3479 (-)/3369 (+) cm(-1) disappeared in the T204A mutant, while being shifted to 3498 (-) and 3474 (+) cm(-1) in the T204S mutant. They appear at 3430 (-)/3402 (+) cm(-1) in the Y174F mutant. From these results, we concluded that the bands at 3479 (-)/3369 (+) cm(-1) originate from the O-H stretch of Thr-204. A stronger hydrogen bond as shown by a large spectral downshift (110 cm(-1)) suggests that the specific hydrogen bonding alteration of Thr-204 takes place upon retinal photoisomerization, which does not occur in the absence of the transducer protein. Thr-204 has been known as an important residue for color tuning and photocycle kinetics in ppR. The results presented here point to an additional important role of Thr-204 in ppR for the interaction with pHtrII. Specific interaction in the complex that involves Thr-204 presumably affects the decay kinetics and binding affinity in the M intermediate.     

Internal water molecules of pharaonis phoborhodopsin studied by low-temperature infrared spectroscopy.

In the Schiff base region of bacteriorhodopsin (BR), a light-driven proton-pump protein, three internal water molecules are involved in a pentagonal cluster structure. These water molecules constitute a hydrogen-bonding network consisting of two positively charged groups, the Schiff base and Arg82, and two negatively charged groups, Asp85 and Asp212. Previous infrared spectroscopy of BR revealed stretching vibrations of such water molecules under strong hydrogen-bonding conditions using spectral differences in D2O and D2(18O) [Kandori and Shichida (2000) J. Am. Chem. Soc. 122, 11745-11746]. The present study extends the infrared analysis to another archaeal rhodopsin, pharaonis phoborhodopsin (ppR; also called pharaonis sensory rhodopsin-II, psR-II), involved in the negative phototaxis of Natronobacterium pharaonis. Despite functional differences between ppR and BR, similar spectral features of water bands were observed before and after photoisomerization of the retinal chromophore at 77 K. This implies that the structure and the structural changes of internal water molecules are similar between ppR and BR. Higher stretching frequencies of the bridged water in ppR suggest that the water-containing pentagonal cluster structure is considerably distorted in ppR. These observations are consistent with the crystallographic structures of ppR and BR. The water structure and structural changes upon photoisomerization of ppR are discussed here on the basis of their infrared spectra.     

Structural changes in the O-decay accelerated mutants of pharaonis phoborhodopsin

pharaonis phoborhodopsin ( ppR, also called pharaonis sensory rhodopsin II, psRII) is a receptor for negative phototaxis in Natronomonas pharaonis. The X-ray crystallographic structure of ppR is very similar to those of the ion-pumping rhodopsins, bacteriorhodopsin (BR) and halorhodopsin (hR). However, the decay processes of the photocycle intermediates such as M and O are much slower than those of BR and hR, which is advantageous for the sensor function of ppR. Iwamoto et al. previously found that, in a quadruple mutant (P182S/P183E/V194T/T204C; denoted as SETC) of ppR, the decay of the O intermediate was accelerated by approximately 100 times ( t 1/2 approximately 6.6 ms vs 690 ms for the wild type of ppR), being almost equal to that of BR (Iwamoto, M., et al. (2005) Biophys. J. 88, 1215-1223). The mutated residues are located on the extracellular surface (Pro182, Pro183, and Val194) and near the Schiff base (Thr204). The present Fourier-transform infrared (FTIR) spectroscopy of SETC revealed that protein structural changes in the K and M states were similar to those of the wild type. In contrast, the ppR O minus ppR infrared difference spectra of SETC are clearly different from those of the wild type in amide-I (1680-1640 cm (-1)) and S-H stretching (2580-2520 cm (-1)) vibrations. The 1673 (+) and 1656 (-) cm (-1) bands newly appear for SETC in the frequency region typical for the amide-I vibration of the alpha II- and alpha I-helices, respectively. The intensities of the 1673 (+) cm (-1) band of various mutants were well correlated with their O-decay half-times. Since the alpha II-helix possesses a considerably distorted structure, the result implies that distortion of the helix is required for fast O-decay. In addition, the characteristic changes in the S-H stretching vibration of Cys204 were different between SETC and T204C, suggesting that structural change near the Schiff base was induced by mutations of the extracellular surface. We conclude that the lifetime of the O intermediate in ppR is regulated by the distorted alpha-helix and strengthened hydrogen bond of Cys204.     

Orientation of the bacteriorhodopsin chromophore probed by polarized Fourier transform infrared difference spectroscopy

Polarized, low-temperature Fourier transform infrared (FTIR) difference spectroscopy has been used to investigate the structure of bacteriorhodopsin (bR) as it undergoes phototransitions from the light-adapted state, bR570, to the K630 and M412 intermediates. The orientations of specific retinal chromophore and protein groups relative to the membrane plane were calculated from the linear dichroism of the infrared bands, which correspond to the vibrational modes of those groups. The linear dichroism of the chromophore C=C and C-C stretching modes indicates that the long axis of the polyene chain is oriented at 20-25 degrees from the membrane plane at 250 K and that it orients more in-plane when the temperature is reduced to 81 K. The polyene plane is found to be approximately perpendicular to the membrane plane from the linear dichroism calculations of the HOOP (hydrogen out-of-plane) wags. The orientation of the transition dipole moments of chromophore vibrations in the K630 and M412 intermediates has been probed, and the dipole moment direction of the C=O bond of an aspartic acid that is protonated in the bR570----M412 transition has been measured.     

FTIR spectroscopy of the K photointermediate of Neurospora rhodopsin: structural changes of the retinal, protein, and water molecules after photoisomerization

Neurospora rhodopsin (NR, also known as NOP-1) is the first rhodopsin of the haloarchaeal type found in eucaryotes. NR demonstrates a very high degree of conservation of the amino acids that constitute the proton-conducting pathway in bacteriorhodopsin (BR), a light-driven proton pump of archaea. Nevertheless, NR does not appear to pump protons, suggesting the absence of the reprotonation switch that is necessary for the active transport. The photocycle of NR is much slower than that of BR, similar to the case of pharaonis phoborhodopsin (ppR), an archaeal photosensory protein. The functional and photochemical differences between NR and BR should be explained in the structural context. In this paper, we studied the structural changes of NR following retinal photoisomerization by means of low-temperature Fourier transform infrared (FTIR) spectroscopy and compared the obtained spectra with those for BR. For the spectroscopic analysis, we established the light-adaptation procedure for NR reconstituted into 1,2-dimyristoyl-sn-glycero- 3-phosphocholine/1,2-dimyristoyl-sn-glycero-3-phosphate (DMPC/DMPA) liposomes, which takes approximately 2 orders of magnitudes longer than in BR. The structure of the retinal chromophore and the hydrogen-bonding strength of the Schiff base in NR are similar to those in BR. Unique spectral features are observed for the S-H stretching vibrations of cysteine and amide-I vibrations for NR before and after retinal isomerization. In NR, there are no spectral changes assignable to the amide bands of alpha helices. The most prominent difference between NR and BR was seen for the water O-D stretching vibrations (measured in D(2)O). Unlike for haloarchaeal rhodopsins such as BR and ppR, no O-D stretches of water under strong hydrogen-bonded conditions (<2400 cm(-1)) were observed in the NR(K) minus NR difference spectra. This suggests a unique hydrogen-bonded network of the Schiff base region, which may be responsible for the lack of the reprotonation switch in NR.     

Vibrational spectrum of the lumi intermediate in the room temperature rhodopsin photo-reaction.

The vibrational spectrum (650-1750 cm(-1)) of the lumi-rhodopsin (lumi) intermediate formed in the microsecond time regime of the room-temperature rhodopsin (RhRT) photoreaction is measured for the first time using picosecond time-resolved coherent anti-Stokes Raman spectroscopy (PTR/CARS). The vibrational spectrum of lumi is recorded 2.5 micros after the 3-ps, 500-nm excitation of RhRT. Complementary to Fourier transform infrared spectra recorded at Rh sample temperatures low enough to freeze lumi, these PTR/CARS results provide the first detailed view of the vibrational degrees of freedom of room-temperature lumi (lumiRT) through the identification of 21 bands. The exceptionally low intensity (compared to those observed in bathoRT) of the hydrogen out-of-plane (HOOP) bands, the moderate intensity and absolute positions of C-C stretching bands, and the presence of high-intensity C==C stretching bands suggest that lumiRT contains an almost planar (nontwisting), all-trans retinal geometry. Independently, the 944-cm(-1) position of the most intense HOOP band implies that a resonance coupling exists between the out-of-plane retinal vibrations and at least one group among the amino acids comprising the retinal binding pocket. The formation of lumiRT, monitored via PTR/CARS spectra recorded on the nanosecond time scale, can be associated with the decay of the blue-shifted intermediate (BSI(RT)) formed in equilibrium with the bathoRT intermediate. PTR/CARS spectra measured at a 210-ns delay contain distinct vibrational features attributable to BSI(RT), which suggest that the all-trans retinal in both BSI(RT) and lumiRT is strongly coupled to part of the retinal binding pocket. With regard to the energy storage/transduction mechanism in RhRT, these results support the hypothesis that during the formation of lumiRT, the majority of the photon energy absorbed by RhRT transfers to the apoprotein opsin.     

Structural change of threonine 89 upon photoisomerization in bacteriorhodopsin as revealed by polarized FTIR spectroscopy.

The all-trans to 13-cis photoisomerization of the retinal chromophore of bacteriorhodopsin occurs selectively, efficiently, and on an ultrafast time scale. The reaction is facilitated by the surrounding protein matrix which undergoes further structural changes during the proton-transporting reaction cycle. Low-temperature polarized Fourier transform infrared difference spectra between bacteriorhodopsin and the K intermediate provide the possibility to investigate such structural changes, by probing O-H and N-H stretching vibrations [Kandori, Kinoshita, Shichida, and Maeda (1998) J. Phys. Chem. B 102, 7899-7905]. The measurements of [3-18O]threonine-labeled bacteriorhodopsin revealed that one of the D2O-sensitive bands (2506 cm(-1) in bacteriorhodopsin and 2466 cm(-1) in the K intermediate, in D2O exhibited 18(O)-induced isotope shift. The O-H stretching vibrations of the threonine side chain correspond to 3378 cm(-1) in bacteriorhodopsin and to 3317 cm(-1) in the K intermediate, indicating that hydrogen bonding becomes stronger after the photoisomerization. The O-H stretch frequency of neat secondary alcohol is 3340-3355 cm(-1). The O-H stretch bands are preserved in the T46V, T90V, T142N, T178N, and T205V mutant proteins, but diminished in T89A and T89C, and slightly shifted in T89S. Thus, the observed O-H stretching vibration originates from Thr89. This is consistent with the atomic structure of this region, and the change of the S-H stretching vibration of the T89C mutant in the K intermediate [Kandori, Kinoshita, Shichida, Maeda, Needleman, and Lanyi (1998) J. Am. Chem. Soc. 120, 5828-5829]. We conclude that all-trans to 13-cis isomerization causes shortening of the hydrogen bond between the OH group of Thr89 and a carboxyl oxygen atom of Asp85.     

Photocycle of phoborhodopsin from haloalkaliphilic bacterium (Natronobacterium pharaonis) studied by low-temperature spectrophotometry.

Phoborhodopsin (pR) is the fourth retinal pigment of Halobacterium halobium and works as a photoreceptor for the negative phototactic response. A similar pigment was previously found in haloalkaliphilic bacterium (Natronbacterium pharaonis) and also works as the receptor of the negative phototactic response; this pigment is called pharaonis phoborhodopsin (ppR). In this paper, the photocycle of ppR was investigated by means of low-temperature spectrophotometry. The absorption maximum of ppR is located at 498 nm, while that of pR is at 487 nm. The absorption spectra of the two have similar vibrational structures. Irradiation of ppR below -100 degrees C produced a K-like intermediate (ppRK) which was a composite of two components. The original ppR and ppRK were perfectly photoreversible. On warming, ppRK was directly converted to an M-like intermediate without formation of the L-like intermediate. The M-like intermediate was converted to the O-like intermediate at pH 7.2, but the O-like intermediate was not detected at pH 9.0. The O-like intermediate then reverted to the original pigment. On the basis of these findings, the photocycle and the primary photochemical process of ppR are presented.     

Structural changes of Salinibacter sensory rhodopsin I upon formation of the K and M photointermediates

Sensory rhodopsin I (SRI) is one of the most interesting photosensory receptors in nature because of its ability to mediate opposite signals depending on light color by photochromic one-photon and two-photon reactions. Recently, we characterized SRI from eubacterium Salinibacter ruber (SrSRI). This protein allows more detailed information about the structure and structural changes of SRI during its action to be obtained. In this paper, Fourier transform infrared (FTIR) spectroscopy is applied to SrSRI, and the spectral changes upon formation of the K and M intermediates are compared with those of other archaeal rhodopsins, SRI from Halobacterium salinarum (HsSRI), sensory rhodopsin II (SRII), bacteriorhodopsin (BR), and halorhodopsin (HR). Spectral comparison of the hydrogen out-of-plane (HOOP) vibrations of the retinal chromophore in the K intermediates shows that extended choromophore distortion takes place in SrSRI and HsSRI, as well as in SRII, whereas the distortion is localized in the Schiff base region in BR and HR. It appears that sensor and pump functions are distinguishable from the spectral feature of HOOP modes. The HOOP band at 864 cm(-1) in SRII, important for negative phototaxis, is absent in SrSRI, suggesting differences in signal transfer mechanism between SRI and SRII. The strongly hydrogen-bound water molecule, important for proton pumps, is observed at 2172 cm(-1) in SrSRI, as well as in BR and SRII. The formation of the M intermediate accompanies the appearance of peaks at 1753 (+) and 1743 (-) cm(-1), which can be interpreted as the protonation signal of the counterion (Asp72) and the proton release signal from an unidentified carboxylic acid, respectively. The structure and structural changes of SrSRI are discussed on the basis of the present infrared spectral comparisons with other rhodopsins.     

Resonance Raman studies of the HOOP modes in octopus bathorhodopsin with deuterium-labeled retinal chromophores

Resonance Raman spectra of the hydrogen out-of-plane (HOOP) vibrational modes in the retinal chromophore of octopus bathorhodopsin with deuterium label(s) along the polyene chain have been obtained. In clear contrast with bovine bathorhodopsin's HOOP modes, there are only two major HOOP bands at 887 and 940 cm-1 for octopus bathorhodopsin. On the basis of their isotopic shifts upon deuterium labeling, we have assigned the band at 887 cm-1 to C10H and C14H HOOP modes, and the band at 940 cm-1 to C11H = C12H Au-like HOOP mode. Except for a 26 cm-1 downward shift, the C11H = C12H Au-like wag appears to be little disturbed in octopus bathorhodopsin from the chromophore in solution since its changes upon deuterium labeling are close to those found in solution model-compound studies. We found also that the C10H and C14H HOOP wags are also similar to those in the model-compound studies. However, we have found that the interaction between the C7H and C8H HOOP internal coordinates of the chromophore in octopus bathorhodopsin is different from that of the chromophore in solution. The intensity of the C11H = C12H and the other HOOP modes suggests that the chromophore of octopus bathorhodopsin is somewhat torsionally distorted from a planar trans geometry. Importantly, a twist about C11 = C12 double bond is inferred. Such a twist breaks the local symmetry, resulting in the observation of the normally Raman-forbidden C11H = C12H Au-like HOOP mode. The twisted nature of the chromophore, semiquantitatively discussed here, likely affects the lambda max of the chromophore and its enthalpy.(ABSTRACT TRUNCATED AT 250 WORDS)     

The M intermediate of Pharaonis phoborhodopsin is photoactive

The retinal protein phoborhodopsin (pR) (also called sensory rhodopsin II) is a specialized photoreceptor pigment used for negative phototaxis in halobacteria. Upon absorption of light, the pigment is transformed into a short-wavelength intermediate, M, that most likely is the signaling state (or its precursor) that triggers the motility response of the cell. The M intermediate thermally decays into the initial pigment, completing the cycle of transformations. In this study we attempted to determine whether M can be converted into the initial state by light. The M intermediate was trapped by the illumination of a water glycerol suspension of phoborhodopsin from Natronobacterium pharaonis called pharaonis phoborhodopsin (ppR) with yellow light (>450 nm) at -50 degrees C. The M intermediate absorbing at 390 nm is stable in the dark at this temperature. We found, however, that M is converted into the initial (or spectrally similar) state with an absorption maximum at 501 nm upon illumination with 380-nm light at -60 degrees C. The reversible transformations ppR if M are accompanied by the perturbation of tryptophan(s) and probably tyrosine(s) residues, as reflected by changes in the UV absorption band. Illumination at lower temperature (-160 degrees C) reveals two intermediates in the photoconversion of M, which we termed M' (or M'(404)) and ppR' (or ppR'(496)). A third photoproduct, ppR'(504), is formed at -110 degrees C during thermal transformations of M'(404) and ppR'(496). The absorption spectrum of M'(404) (maximum at 404 nm) consists of distinct vibronic bands at 362, 382, 404, and 420 nm that are different from the vibronic bands of M at 348, 368, 390, and 415 nm. ppR'(496) has an absorption band that is shifted to shorter wavelengths by 5 nm compared to the initial ppR, whereas ppR'(504) is redshifted by at least 3 nm. As in bacteriorhodopsin, photoexcitation of the M intermediate of ppR and, presumably, photoisomerization of the chromophore during the M --> M' transition result in a dramatic increase in the proton affinity of the Schiff base, followed by its reprotonation during the M' --> ppR' transition. Because the latter reaction occurs at very low temperature, the proton is most likely taken from the counterion (Asp(75)) rather than from the bulk. The phototransformation of M reveals a certain heterogeneity of the pigment, which probably reflects different populations of M or its photoproduct M'. Photoconversion of the M intermediate provides a possible pathway for photoreception in halobacteria and a useful tool for studying the mechanisms of signal transduction by phoborhodopsin (sensory rhodopsin II).