The photocycle of a flavin-binding domain of the blue light photoreceptor phototropin.

The plant blue light receptor, phot1, a member of the phototropin family, is a plasma membrane-associated flavoprotein that contains two ( approximately 110 amino acids) flavin-binding domains, LOV1 and LOV2, within its N terminus and a typical serine-threonine protein kinase domain at its C terminus. The LOV (light, oxygen, and voltage) domains belong to the PAS domain superfamily of sensor proteins. In response to blue light, phototropins undergo autophosphorylation. E. coli-expressed LOV domains bind riboflavin-5'-monophosphate, are photochemically active, and have major absorption peaks at 360 and 450 nm, with the 450 nm peak having vibronic structure at 425 and 475 nm. These spectral features correspond to the action spectrum for phototropism in higher plants. Blue light excitation of the LOV2 domain generates, in less than 30 ns, a transient approximately 660 nm-absorbing species that spectroscopically resembles a flavin triplet state. This putative triplet state subsequently decays with a 4-micros time constant into a 390 nm-absorbing metastable form. The LOV2 domain (450 nm) recovers spontaneously with half-times of approximately 50 s. It has been shown that the metastable species is likely a flavin-cysteine (Cys(39) thiol) adduct at the flavin C(4a) position. A LOV2C39A mutant generates the early photoproduct but not the adduct. Titrations of LOV2 using chromophore fluorescence as an indicator suggest that Cys(39) exists as a thiolate.

Multicolored protein conformation states in the photocycle of transducer-free sensory rhodopsin-I.

Sensory rhodopsin-I (SRI), a phototaxis receptor of archaebacteria, is a retinal-binding protein that exists in the cell membrane intimately associated with a signal-transducing protein (HtrI) homologous to eubacterial chemotaxis receptors. Transducer-free sensory rhodopsin-I (fSRI), from cells devoid of HtrI, undergoes a photochemical cycle kinetically different from that of native SRI. We report here on the measurement and analysis of the photochemical kinetics of fSRI reactions in the 350-750-nm spectral range and in a 10(-7) s to 1 s time window. The lack of specific intermolecular interactions between SRI and HtrI results in early return of the ground form via distinct branching reactions in fSRI, not evident in the photocycle of native SRI. The chromophore transitions are loosely coupled to protein structural transitions. The coexistence of multiple spectral forms within kinetic intermediates is interpreted within the concept of multicolored protein conformational states.

New photointermediates in the two photon signaling pathway of sensory rhodopsin-I.

Sensory rhodopsin-I (SRI) functions as a color discriminating receptor in halobacterial phototaxis. SRI exists in the membrane as a molecular complex with a signal transducer protein. Excitation of its thermally stable form, SRI(587), generates a long-lived photointermediate of its photocycle, S(373), and an attractant phototactic response. S(373) decays thermally in a few seconds into SRI(587.) However, when S(373) is excited by UV-blue light, it photoconverts into SRI(587) in less than a second, generating a repellent phototactic response. Only one intermediate of this back-photoreaction, S(b)(510), is known. We studied the back-photoreaction in both native SRI and its transducer free form fSRI by measuring laser flash induced absorption changes of S(373) photoproducts from 100 ns to 1 s in the 350-750 nm range. Using global exponential fitting, we determined the spectra and kinetics of the photointermediates. S(373) and fS(373) when pumped with 355 nm laser light generate in less than 100 ns two intermediate species: a previously undetected species that absorbs maximally at about 410 nm, S(b)(410), and the previously described S(b)(510). These two intermediates appear to be in a rapid equilibrium, which probably entails protonation change of the Schiff base chromophore. At pH 6 this system relaxes to SRI(587) via another intermediate absorbing maximally around 550 nm, which thermally decays back to the ground state. The same intermediates are seen in the presence and absence of transducer; however, the kinetics are affected by binding of the transducer.

Reversal of blue light-stimulated stomatal opening by green light.

Blue light-stimulated stomatal opening in detached epidermis of Vicia faba is reversed by green light. A 30 s green light pulse eliminated the transient opening stimulated by an immediately preceding blue light pulse. Opening was restored by a subsequent blue light pulse. An initial green light pulse did not alter the response to a subsequent blue light pulse. Reversal also occurred under continuous illumination, with or without a saturating red light background. The magnitude of the green light reversal depended on fluence rate, with full reversal observed at a green light fluence rate twice that of the blue light. Continuous green light given alone stimulated a slight stomatal opening, and had no effect on red light-stimulated opening. An action spectrum for the green light effect showed a maximum at 540 nm and minor peaks at 490 and 580 nm. This spectrum is similar to the action spectrum for blue light-stimulated stomatal opening, red-shifted by about 90 nm. The carotenoid zeaxanthin has been implicated as a photoreceptor for the stomatal blue light response. Blue/green reversibility might be explained by a pair of interconvertible zeaxanthin isomers, one absorbing in the blue and the other in the green, with the green absorbing form being the physiologically active one.

The ultraviolet action spectrum for stomatal opening in broad bean.

The ultraviolet action spectrum for stomatal opening was measured using epidermal peels from leaves of broad bean (Vicia faba). The spectrum was calculated from hyperbolic fluence response curves using 11 wavelengths ranging from 275 to 459 nm. The action spectrum exhibits a major peak at approximately 280 nm and a minor peak at approximately 360 nm. The response at 280 nm is about three times greater than the response at 459 nm. Under the conditions utilized (i.e. the absence of saturating red light), stomatal opening saturated at extremely low fluence rates: <0.2 micromol m(-2) s(-1) at 280 nm, and approximately 1.0 micromol m(-2) s(-1) at 459 nm. The threshold for blue-light-induced stomatal opening was approximately 0.02 micromol m(-2) s(-1). In light-mixing experiments, the addition of 280 nm light to saturating 650 nm (red) light caused additional stomatal opening, which is indicative of separate photoreceptors. In contrast, adding 280 nm of light to saturating 459 nm (blue) light did not increase stomatal opening, suggesting that they both excite the same receptor. The results with white light were similar to those with blue light. We infer that ultraviolet light acts via the blue light photoreceptor rather than through photosynthesis. The additional absorbance peak at 360 nm suggests that the chromophore is either a flavin or a cis-carotenoid, both of which exhibit peaks in this region. It is proposed that the chromophore can be excited either directly by blue light or by energy transferred from the protein portion of the protein-pigment complex after it absorbs 280 nm light.

The photocycle of bacteriorhodopsin at high pH and ionic strength. I. Effects of pH and buffer on the absorption kinetics.

A fitting analysis resolved the kinetics in the microsecond to second time range of the absorption changes in the bacteriorhodopsin photocycle at pH = 8.0-9.5 in 3 M KCl into seven exponential components. The time constants and/or amplitudes of all components are strongly pH-dependent. In the pH range studied, the logarithms of the pH-dependent time constants varied linearly with pH. The maximum absolute value of the corresponding slopes was 0.4, in contrast with the theoretically expected value of 1 for unidirectional reactions coupled directly to proton exchange with the bulk phase. This indicates that the extracted macroscopic rate constants are not identical to the microscopic rate constants for the elementary photocycle reaction steps. Unexpected differences were found in the kinetic parameters in CHES and borate buffers.

The photocycle of bacteriorhodopsin at high pH and ionic strength. II. Time-dependent anisotropy studied by partially saturating photoselection.

Photoselection measurements with moderate excitation intensity on bacteriorhodopsin (bR) immobilized in a polyacrylamide gel soaked in 3 M KCl in the pH range 8.0-9.5 resulted in an unusual time-dependent anisotropy. In the microsecond region, the anisotropy exhibits a constant level that is considerably less than 2/5 theoretically expected for the vanishing excitation intensity, indicating partial saturation. In the millisecond region, it becomes time-dependent. Theoretical models for such a time-dependent anisotropy are presented. These models include a consideration of: (i) reorientation of the retinal chromophore during or after excitation, (ii) parallel reactions of differently saturated photoselected species of a heterogenous bR population preexisting in the ground state or photochemically induced, (iii) branching in a photochemical step, and (iv) cooperativity of molecules within a trimer. All of these models describe the anisotropy as a ratio of sums of exponentials, where the rate constants correspond to the kinetics of the photocycle. An analysis of the fitted amplitudes of the exponentials favors the models involving parallel processes rather than those invoking chromophore reorientation.

Chromophore reorientations in the early photolysis intermediates of bacteriorhodopsin.

The photoselection-induced time-resolved linear dichroism of a bacteriorhodopsin suspension of purple membrane from 350 to 750 nm is measured by a new pseudo-null measurement technique. In combination with time-resolved absorption measurements, these linear dichroism measurements are used to determine the reorientation of the retinal chromophore of bacteriorhodopsin from 50 ns to 50 microseconds after photolysis. This time range covers the times when the K photointermediate decays to form L, as well as the early times during the formation of the M intermediate in the photocycle. An analysis of the photoselection-induced linear dichroism measured directly, along with the absorbance changes polarized parallel to the linearly polarized excitation, shows that the anisotropy is invariant over this time period, implying that the photolyzed chromophore rotates less than 8 degrees C with respect to unphotolyzed chromophores during this part of the photocycle.

Removal of transducer HtrI allows electrogenic proton translocation by sensory rhodopsin I.

Sensory rhodopsin I (sR-I) is a phototaxis receptor in halobacteria, which is closely related to the light-driven proton pump bacteriorhodopsin and the chloride pump halorhodopsin found in the same organisms. The three pigments undergo similar cyclic photoreactions, in spite of their different functions. In intact cells or isolated membranes sR-I is complexed with protein HtrI, the next link in the signal transduction chain, and does not function as an electrogenic ion pump. However, illumination of sR-I in membranes lacking HtrI causes pH changes in the medium, and its photoreaction kinetics become pH-dependent. We show here that in closed vesicles, near neutral pH it functions as an electrogenic proton pump capable of generating at least -80 mV transmembrane potential. The action spectrum shows a maximum 37 nm below the 587-nm absorption maximum of the native pigment. This apparent discrepancy occurs because the 587-nm form of HtrI-free sR-I exists in a pH-dependent equilibrium with a 550-nm absorbing species generated through deprotonation of one group with a pKa of 7.2, which we have tentatively identified as Asp-76. We interpret the results in terms of a general model for ion translocation by the bacterial rhodopsins.

Picosecond dynamics of bacteriorhodopsin, probed by time-resolved infrared spectroscopy.

The photoinduced reaction cycle of bacteriorhodopsin (BR) has been studied by means of a recently developed picosecond infrared spectroscopic method at ambient temperature. BR - K difference spectra between 1560 and 1700 cm-1 have been recorded at delay times from 100 ps to 14 ns. The spectrum remains unchanged during this period. The negative difference OD band at 1660 cm-1 indicates the peptide backbone responds within 50 ps. A survey in the region of carboxylic side chain absorption around 1740 cm-1 reveals that perturbations of those groups, present in low-temperature FTIR spectra, are not observable within 10 ns, suggesting a slow conformational change.

Sensory rhodopsin I: receptor activation and signal relay.

Recent progress is summarized on the mechanism of phototransduction by sensory rhodopsin I (SR-I), a phototaxis receptor in Halobacterium halobium. Two aspects are emphasized: (i) The coupling of retinal isomerization to protein conformational changes. Retinal analogs have been used to probe chromophore-apoprotein interactions during the receptor activation process. One of the most important results is the finding of a steric trigger deriving from the interaction of residues on the protein with a methyl group near the isomerizing bond of the retinal (at carbon 13). Recent work on molecular genetic methods to further probe structure/function includes the synthesis and expression of an SR-I apoprotein gene designed for residue replacements by cassette mutagenesis, and transformation of an H. halobium mutant lacking all retinylidene proteins known in this species to SR-I+ and bacteriorhodopsin (BR)+. (ii) The relay of the SR-I signal to a post-receptor component. A carboxylmethylated protein ("MPP-I") associated with SR-I and found in the H. halobium membrane exhibits homology with the signaling domain of eubacterial chemotaxis transducers (e.g., Escherichia coli Tar, Tsr, and Trg proteins), suggesting a model based on SR-I----MPP-I signal relay.

Structure of the retinal chromophore in sensory rhodopsin I from resonance Raman spectroscopy.

Sensory rhodopsin I (SR-I) is a retinal-containing pigment which functions as a phototaxis receptor in Halobacterium halobium. We have obtained resonance Raman vibrational spectra of the native membrane-bound form of SR587 and used these data to determine the structure of its retinal prosthetic group. The similar frequencies and intensities of the skeletal fingerprint modes in SR587, bacteriorhodopsin (BR568), and halorhodopsin (HR578) as well as the position of the dideuterio rocking mode when SR-I is regenerated with 12,14-D2 retinal (915 cm-1) demonstrate that the retinal chromophore has an all-trans configuration. The shift of the C = N stretching mode from 1628 cm-1 in H2O to 1620 cm-1 in D2O demonstrates that the chromophore in SR587 is bound to the protein by a protonated Schiff base linkage. The small shift of the 1195 cm-1 C14-C15 stretching mode in D2O establishes that the protonated Schiff base bond has an anti configuration. The low value of the Schiff base stretching frequency together with its small 8 cm-1 shift in D2O indicates that the Schiff base proton is weakly hydrogen bonded to its protein counterion. This suggests that the red shift in the absorption maximum of SR-I (587 nm) compared with HR (578 nm) and BR (568 nm) is due to a reduction of the electrostatic interaction between the protonated Schiff base group and its protein counterion.

Halorhodopsin and sensory rhodopsin contain a C6-C7 s-trans retinal chromophore.

Halorhodopsin (HR) and sensory rhodopsin (SR) have been regenerated with retinal analogues that are covalently locked in the 6-s-cis or 6-s-trans conformations. Both pigments regenerate more completely with the locked 6-s-trans retinal and produce analogue pigments with absorption maxima (577 nm for HR and 592 nm for SR) nearly identical to those of the native pigments (577 and 587 nm). This indicates that HR and SR bind retinal in the 6-s-trans conformation. The opsin shift for the locked 6-s-trans analogue in HR is 1,200 cm-1 less than that for the native chromophore (5,400 cm-1). The opsin shift for the 6-s-trans analogue in SR is 1,100 cm-1 less than that for the native retinal (5,700 cm-1). This demonstrates that approximately 20% of the opsin shift in these pigments arises from a protein-induced change in the chromophore conformation from twisted 6-s-cis in solution to planar 6-s-trans in the protein. The reduced opsin shift observed for the locked 6-s-cis analogue pigments compared with the locked 6-s-trans pigments may be due to a positive electrostatic perturbation near C7.

The photochemical reactions of bacterial sensory rhodopsin-I. Flash photolysis study in the one microsecond to eight second time window.

Halobacterium halobium Flx mutants are deficient in bacteriorhodopsin (bR) and halorhodopsin (hR). Such strains are phototactic and the light signal detectors are two additional retinal pigments, sensory rhodopsins I and II (sR-I and sR-II), which absorb maximally at 587 and 480 nm, respectively. A retinal-deficient Flx mutant, Flx5R, overproduces sR-I-opsin and does not show any photochemical activity other than that of sR-I after the pigment is regenerated by addition of all-trans retinal. Using native membrane vesicles from this strain, we have resolved a new photointermediate in the sR-I photocycle between the early bathointermediate S610 and the later intermediate S373. The new form, S560, resembles the L intermediate of bR in its position in the photoreaction cycle, its relatively low extinction, and its moderate blue shift. It forms with a half-time of approximately 90 microseconds at 21 degrees C, concomitant with the decay of S610. Its decay with a half-time of 270 microseconds parallels the appearance of S373. From a data set consisting of laser flash-induced absorbance changes (300 ns, 580-nm excitation) measured at 24 wavelengths from 340 to 720 nm in a time window spanning 1 microsecond to 8 s we have calculated the spectra of the photocycle intermediates assuming a unidirectional, unbranched reaction scheme.

Tyrosine and carboxyl protonation changes in the bacteriorhodopsin photocycle. 2. Tyrosines-26 and -64.

Low-temperature Fourier transform infrared (FTIR) and UV difference spectroscopies combined with selective tyrosine nitration and tyrosine isotopic labeling have been used to investigate the participation of tyrosines-26 and -64 in the bacteriorhodopsin (bR) photocycle. Nitration of Tyr-26 has no detectable effect on the FTIR or UV difference spectra of the BR570----K630 or BR570----M412 transitions. In contrast, nitration of Tyr-64 causes changes in both the FTIR and UV spectra of these transitions. However, this nitration does not alter tyrosine peaks in the FTIR difference spectra which have previously been associated with the protonation of a tyrosinate by K630 and the deprotonation of a tyrosine by M412 [Roepe, P., Ahl, P. L., Das Gupta, S. K., Herzfeld, J., & Rothschild, K. J. (1987) Biochemistry (preceding paper in this issue)]. Instead, Tyr-64 nitration appears to affect other tyrosine peaks. These results and changes in UV difference spectra upon Tyr-64 nitration are consistent with the deprotonation of Tyr-64 by M412 as concluded previously [Scherrer, P., & Stoeckenius, W. (1985) Biochemistry 24, 7733-7740]. Effects on chromophore vibrations caused by Tyr-64 nitration are unaltered upon reducing the nitrotyrosine to aminotyrosine with sodium dithionite. Finally, nitro-Tyr-64 causes a shift in the frequency of a positive peak at 1739 cm-1 in the BR570----M412 FTIR difference spectrum which reflects the protonation of a carboxyl-containing residue [Engelhard, M., Gerwert, K., Hess, B., Kreutz, W., & Siebert, F. (1985) Biochemistry 24, 400-407; Roepe, P., Ahl, P. L., Das Gupta, S. K., Herzfeld, J., & Rothschild, K. J. (1987) Biochemistry (preceding paper in this issue)]. The shift does not occur for samples containing amino-Tyr-64. These data suggest that Tyr-64 may interact with this carboxyl group.

Structure of the retinal chromophore in the hRL intermediate of halorhodopsin from resonance raman spectroscopy.

Time-resolved resonance Raman spectra of the hRL intermediate of halorhodopsin have been obtained. The structurally sensitive fingerprint region of the hRL spectrum is very similar to that of bacteriorhodopsin's L550 intermediate, which is known to have a 13-cis configuration. This indicates that hRL contains a 13-cis chromophore and that an all-trans----13-cis isomerization occurs in the halorhodopsin photocycle. hRL exhibits a Schiff base stretching mode at 1644 cm-1, which shifts to 1620 cm-1 in D2O. This demonstrates that the Schiff base linkage to the protein is protonated. The insensitivity of the C-C stretching mode frequencies to N-deuteriation suggests that the Schiff base configuration is anti. The 24 cm-1 shift of the Schiff base mode in D2O indicates that the Schiff base proton in hRL has a stronger hydrogen-bonding interaction with the protein than does hR578.

Biochemical and spectroscopic characterization of the blue-green photoreceptor in Halobacterium halobium.

Spectroscopic evidence indicates the presence of a second sensory receptor sR-II in Halobacterium halobium, which causes a repellent response to blue-green light. Reactions with hydroxylamine and NaCNBH3 and reconstitution of the bleached pigment with retinal show that it is very similar to the other retinylidene pigments bacteriorhodopsin, halorhodopsin, and especially the earlier-discovered phototaxis receptor, sensory rhodopsin, renamed sR-I587. The second sensory receptor, sR-II480, has an absorbance maximum at 480 nm and undergoes a cyclic photoreaction with a half-time of approximately 200 msec. Its predominant photocycle intermediate absorbs maximally near 360 nm. The receptor can be detected spectroscopically in the presence of sR-I587 and quantitated through its transient response to 450-nm excitation. It is selectively bleached by low hydroxylamine concentrations that are insufficient to bleach sR-I587 significantly. Its photochemical and phototactic activities can be restored by addition of retinal. The mobility of the receptor, on NaDodSO4/polyacrylamide gels, was similar or identical to that of sR-I587 and slightly faster than bacteriorhodopsin, yielding an apparent molecular mass of 23-24 kDa.

Color discrimination in halobacteria: spectroscopic characterization of a second sensory receptor covering the blue-green region of the spectrum.

Halobacterium halobium is attracted by green and red light and repelled by blue-green and shorter wavelength light. a photochromic, rhodopsin-like protein in the cell membrane, sensory rhodopsin sR587, has been identified as the receptor for the long-wavelength and near-UV stimuli. Discrepancies between the action spectrum for the repellent effect of blue light and the absorption spectrum of sR587 and its photocycle intermediate S373 strongly suggest the existence of an additional photoreceptor for the blue region of the spectrum. Transient light-induced absorbance changes in intact cells and cell membranes show, in addition to sR587, the presence of a second photoactive pigment with maximal absorption near 480 nm. It undergoes a cyclic photoreaction with a half-time of 150 msec. One intermediate state with maximal absorption near 360 nm has been resolved. The spectral properties of the new pigment are consistent with a function as the postulated photoreceptor for the repellent effect of blue light. The phototactic reactions and both pigments are absent when retinal synthesis is blocked; both can be restored by the addition of retinal. These results confirm and extend similar observations by Takahashi et al. [Takahashi, T., Tomioka, H., Kamo, N. & Kobatake, Y. (1985) FEMS Microbiol. Lett. 28, 161-164]. The archaeobacterium H. halobium thus uses two different mechanisms for color discrimination; it uses two rhodopsin-like receptors with different spectral sensitivities and also the photochromicity of at least one of these receptors to distinguish between three regions covering the visible and near-UV spectrum.

Chromophore/protein interaction in bacterial sensory rhodopsin and bacteriorhodopsin.

Retinal analogues with altered conjugated double bond systems or altered stereochemistry were incorporated into the phototaxis receptor sensory rhodopsin (SR) and the light-driven proton pump bacteriorhodopsin (BR) from Halobacterium halobium. Wavelength shifts in absorption ("opsin shifts") due to analogue interaction with the protein microenvironment demonstrate that the same overall electrostatic and steric properties of the retinal binding-site structures exist in both proteins despite their different functions. pi-Electron calculations from the opsin shifts lead to a new description of protein charge distribution that applies to the binding sites of both SR and BR. The new data extends the previously proposed external point charge model for BR to include an ion-pair protein/chromophore interaction near the beta-ionone moiety. The new data modifies the previously proposed external point-charge model, the derivation of which involved an experimentally erroneous opsin shift for one of the BR analogues.