INTRODUCTION

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Some species of Archaea, bacteria and unicellular flagellated algae, have developed a light-induced motile response, known as phototaxis, of reacting to changes in the light environment and migrating to optimum light conditions. In Halobacteria, the light-driven proton pump bacteriorhodopsin (Oesterhelt and Stoeckenius, 1971) was shown to mediate motile responses in cells, presumably through light-induced changes of the transmembrane potential as a light is turned on and off (Yan et al., 1992; Bibikov et al., 1993). In addition to bacteriorhodopsin, Halobacteria developed three other specialized retinal proteins structurally related to bacteriorhodopsin (Oesterhelt, 1998): halorhodopsin, a light-driven chloride pump (Matsuno-Yagi and Mukohata, 1977; Lanyi, 1990); sensory rhodopsin I (sRI) (Bogomolni and Spudich, 1982); and phoborhodopsin (Takahashi et al., 1985b; Tomioka et al., 1986) also called sensory rhodopsin II (sRII) (Wolff et al., 1986; Marwan and Oesterhelt, 1987). The photophobic receptor of Natronobacterium pharaonis is closely related (Scharf et al., 1992) to the phoborhodopsin of Halobacterium salinarum (former halobium) and is called pharaonis phoborhodopsin (ppR) or pharaonis sensory rhodopsin II (psRII). Signal transduction in these specialized photoreceptors occurs through the activation of transducer proteins. These proteins are activated after phototransformation of sensory rhodopsins into their active signaling state (see reviews in Spudich et al., 1995; Hoff et al., 1997). This type of signal transduction apparently enables a bacterium to achieve a higher sensitivity compared to phototaxis mediated by bR. Sensory rhodopsins undergo cyclic photochemical transformations (Spudich and Bogomolni, 1988; Miyazaki et al., 1992; Chizhov et al., 1998) that are similar in many aspects to those of bacteriorhodopsin, including the formation of intermediates analogous to the intermediates of bR, K, L, M, N, and O (in sRI the last two states have not been detected). In both pigments the signaling state is formed upon light-induced deprotonation of the Schiff base, leading to formation of the M intermediate, which absorbs in the deep violet/near-UV. The M intermediate slowly (in seconds) thermally converts into the initial state.

The functions of the two photoreceptor pigments are different; sRI is maximally sensitive to orange light (590 nm), which produces an attractant reaction. The attraction to light facilitates the accumulation of cells in the light, which provides the energy that can drive proton transport by bR and chloride transport by halorhodopsin. Photoexcitation of the M intermediate of sRI, which absorbs at 370 nm, results in a photophobic response (Spudich and Bogomolni, 1984).

The function of phoborhodopsin (sensory rhodopsin II) is primarily to detect and help to avoid exposure to potentially harmful short-wavelength light (maximum absorbance at 480-490 nm) (Takahashi et al., 1985b; Wolff et al., 1986; Marwan and Oesterhelt, 1987; Hoff et al., 1997). Absorption of a light quantum by phoborhodopsin causes reversal of the direction of swimming (photophobic reaction). It was suggested that the M intermediate (as well as O) might be a signaling state of the pigment (Yan et al., 1991). As in the case of bacteriorhodopsin, in ppR the formation of the M intermediate involves the all-trans to 13-cis photoisomerization of the chromophore (Imamoto et al., 1992a), which initiates proton transport from the Schiff base to a nearby aspartic acid, Asp⁷⁵ (Engelhard et al., 1996). The decay of M is slowed compared to the bR photocycle, presumably because of the absence of an efficient proton donor analogous to Asp⁹⁶ (Iwamoto et al., 1999a). The decay of M can be accelerated by the addition of azide, which is similar to that in the D96N mutant of bacteriorhodopsin (Takao et al., 1998). M decay and formation of the O intermediate are accompanied by proton uptake, whereas O decay correlates with proton release to the bulk in sRII and ppR (Sasaki and Spudich, 1999; Iwamoto et al., 1999b). Sasaki and Spudich observed that net proton release and uptake occurred at the same (extracellular) side of the membrane in H. salinarum envelop vesicles under their conditions, and therefore little or no transmembrane proton transport occurred. Iwamoto et al. (1999b) suggested, on the other hand, that in their conditions proton uptake might occur from the cytoplasmic surface and proton release to the extracellular surface of ppR.

The photocycle of phoborhodopsin has been studied both by low-temperature steady-state (Imamoto et al., 1991; Hirayama et al., 1992) and room-temperature kinetic spectroscopy (Imamoto et al., 1992b; Miyazaki et al., 1992; Chizhov et al., 1998). However, it has not been established whether the M intermediate of phoborhodopsin is photoactive, and if it is, what the pathway and physiological response of its phototransformation are. Investigation of this reaction will provide a useful approach to study the mechanism of the photophobic response and its connection with the intramolecular proton transfer reactions.

In bacteriorhodopsin, photoexcitation of the M intermediate causes fast reprotonation of the Schiff base (Litvin et al., 1975; Druckmann et al., 1992) from the counterion Asp⁸⁵ (Balashov and Litvin, 1981a; Takei et al., 1992) and transformation of the pigment back to its initial state through a nonpumping pathway (Karvaly and Dancshazy, 1977; Ormos et al., 1978; Litvin et al., 1981; Ludmann et al., 1999). Phototransformation of M (reviewed by Balashov, 1995) involves the formation of two primary photoproducts, P421 and P433 (Litvin and Balashov, 1977; Balashov and Litvin, 1981b), called also M' (Hurley et al., 1978), and several subsequent thermal intermediates, P565, P575, and P585 (or bR'), which are in turn photoactive (Balashov and Litvin, 1981a; Balashov et al., 1988).

In the case of sRI, the phototransformation of the M intermediate is utilized to provide a second transient signaling pigment (Spudich and Bogomolni, 1984; Hoff et al., 1997; Takahashi et al., 1985a): light absorption by the initial state (maximum at 587 nm) produces a positive motile response, whereas absorption of light by its photointermediate M (maximum at 373 nm) results in a negative (photophobic) response.

In this study we examined the photoactivity of the M intermediate of ppR at low temperatures, using ppR expressed in Escherichia coli and solubilized in a detergent (Shimono et al., 1997; Iwamoto et al., 1999b).

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Expression of ppR in E. coli was detected as described earlier (Shimono et al., 1997). The pigment was purified as described by Iwamoto et al. (1999). Stock suspension contained pigment in 0.5% n-dodecyl--D-maltoside (DM), 100 mM KCl, and 50 mM Tris buffer (pH 8.0). For low-temperature measurements it was diluted with two parts glycerol. The final pH was 7 (at 25°C). A homemade cryostat enabled us to perform measurements at temperatures from 25°C to 160°C in the 250-750-nm spectral range. The pathlength of the cuvette was 2 mm. Spectra were recorded on a Cary-Aviv 14DS UV-VIS spectrophotometer. Bandwidth of the monochromator was 1 nm. Spectra were recorded in 1-nm or 0.5-nm steps. The latter was mainly used to resolve the light-induced changes in the UV. In addition to the conventional methods of low-temperature absorption spectroscopy, we also used derivative spectroscopy. The second derivative (d²A/d²) spectra significantly improved the resolution of overlapping vibrational bands (Balashov et al., 1991b). They are also helpful in the detection of small but narrow bands because the intensity of a band in the second derivative spectrum is inversely proportional to the square of the half-width of the absorption band. Application of the second derivative technique enabled us to easily determine the fraction of ppR in the photosteady-state mixture with its bathoproduct K and to resolve the vibrational structure of the absorption spectra of ppR and its photoproducts.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The M intermediate was formed upon illumination of ppR with 450-550-nm light at 50°C. About 50% of the pigment was transformed into M under these conditions (Fig. 1 A, curves 1 and 2). A larger amount of the pigment can be converted into M upon illumination of the sample at 450-550 nm while the temperature is decreased from 20°C to 50°C. However, a small fraction of other intermediates may be trapped along with M under these conditions, particularly an O-like species absorbing around 560 nm. Therefore we used illumination at 50°C to form M. 

View larger version (17K): [in this window] [in a new window]   FIGURE 1   Absorption changes accompanying the formation and photoconversion of the M intermediate of ppR. (A) Absorption spectra measured at 60°C: (1) initial ppR; (2) after 5 min of illumination at 450-550 nm at 50°C to convert ppR into M and cooling to 60°C; 3, after 5 min of illumination at 360-420 nm at 60°C. (B) Difference spectra of formation (1) and photoconversion (2) of M. Spectra 1 and 2 were obtained, respectively, as the difference spectrum 2 minus 1 and spectrum 3 minus 2 of A. (C) Resolution of the vibronic bands of ppR and M at 60°C using the second derivative: d2A/d2 of the difference spectrum of transformation of initial ppR into M at 60°C (of spectrum 1 in B).

The absorption maximum of ppR is at 501 nm at 60°C, whereas M has its maximum at 390 nm in the M minus ppR difference spectrum (Fig. 1 B). The difference spectrum of M minus ppR at 60°C indicates that transformation of ppR into M is accompanied by an increase in the intensity of the 296-nm and 286-nm bands and a decrease of absorbance around 260-270 nm (Fig. 1 B, curve 1, and Fig. 2, A and B, curve 1). Interestingly, the changes in the UV accompanying formation of the M intermediate in ppR were different from those observed upon formation of the M intermediate in bacteriorhodopsin (Fig. 2 B, compare curves 1 and 2).

View larger version (18K): [in this window] [in a new window]   FIGURE 2   (A) Comparison of the absorption changes accompanying the formation of the M intermediate in ppR (curve 1) and bR (curve 2) at 50°C. Both samples contained 30 mM KCl and 15 mM Tris-buffer (pH 7.0). The difference spectrum of the bR to M transition in UV is similar to the earlier published spectrum (Sabés et al., 1984). (B) The UV region of the difference spectra accompanying (1) the formation of the M intermediate in ppR (Fig. 1 B, curve 1), (2) the formation of the M intermediate in bR at 50°C, and (3) photoconversion of the M intermediate in ppR (Fig. 1 B, curve 2).

The vibrational structure of ppR and its M photoproduct can be resolved using the second derivative of the M minus ppR difference absorption spectrum (Fig. 1 C). Transformation of ppR into M was accompanied by a decrease in the 503-nm and 462-nm vibronic bands of ppR and the formation of 415-, 390-, 368-, and 348-nm vibronic bands of M (Fig. 1 C). In the UV region several peaks appeared in the second derivative spectrum at 296, 286, 278, 266, and 258 nm, which were caused by perturbation of aromatic residues in the ppR  M transition.

Photoconversion of M at 60°C and at 160°C: formation of photointermediates M' and ppR'

The M intermediate is stable at 60°C in the dark (absorption spectrum does not change upon incubation for 30 min in the dark at 60°C). However, illumination of a sample containing M at 60°C with 360-420-nm light caused a transformation of most of M back to the pigment's initial state or a spectrally isochromic state (Fig. 1 A, curve 3), indicating that M is photoactive. The absorption maximum of the pigment photoregenerated from M at 60°C (Fig. 1 B, curve 2) is at 501 nm. It practically coincides with the maximum of the initial pigment. The absorption changes in the UV were reversed upon photoconversion of M back to ppR at 60°C (Fig. 2 B, curve 3).

Experiments at lower temperature (160°C) revealed intermediates in the photoconversion of M. Illumination of the sample containing M at 160°C (Fig. 3 A, curve 1) resulted in a transformation of M into a long-wavelength species (Fig. 3 A, curve 2). This indicates that light-induced protonation of the Schiff base takes place, even at 160°C. The absorption maximum of the photoproduct in the difference spectrum (Fig. 3 B, curve 1) is at 496 nm; it is blueshifted 5 nm compared to the maximum of the initial ppR at 160°C (501 nm). We call this species ppR'₄₉₆.

View larger version (17K): [in this window] [in a new window]   FIGURE 3   Photoconversion of the M intermediate of ppR at 160°C. (A) (1) Spectrum containing ~60% M and 40% ppR. (2) After 5 min of illumination at 360-420 nm at 160°C. (3) After 5 min of illumination at 450-550 nm. (B) Difference spectra of (1) photoconversion of M at 160°C (spectrum 2  spectrum 1 in A); (2) photoconversion of ppR' into M (spectrum 3  spectrum 2). (C) Minus second derivative of the difference spectrum of photoconversion of ppR' at 160°C (d2/d2 of spectrum 2 in B).

The photoproduct ppR'₄₉₆ differs from the initial ppR not only in its blueshifted spectrum, but also in its photochemical properties. Illumination of ppR'₄₉₆ at 160°C resulted in a conversion of the main fraction of ppR'₄₉₆ back to M (Fig. 3 A, curve 3). The second derivative of the difference spectrum clearly showed minimum at 496 nm and characteristic bands of M at 414, 390, and 369 nm (Fig. 3 C) due to the ppR'₄₉₆  M photoconversion.

The initial ppR at the same temperature (160°C) undergoes phototransformation into its bathoproduct K (Fig. 4 A). From the amplitude of vibronic bands in the initial ppR and the photo-steady-state mixture of ppR and K produced by 460-nm light (Fig. 4 B), the fraction of K (52 ± 2%) and the ratio of the quantum yield of the forward and back reactions (₁/₂ = 0.7 ± 0.1) can be estimated. The values obtained are similar to those for the primary light reaction in bacteriorhodopsin (Balashov et al., 1991b).

View larger version (20K): [in this window] [in a new window]   FIGURE 4   Phototransformation of the initial ppR at 160°C. (A) Absorption spectra (1) of ppR; (2) after illumination for 15 min at 460 nm; (3) of calculated spectrum of the bathoproduct K, assuming that 52% of ppR is transformed into K upon illumination with 460-nm light. (B) Curves 1 and 2, minus second derivatives of spectra 1 and 2 of A, respectively. From the decrease of the amplitudes of 503-nm and 460-nm vibronic bands of ppR after illumination, the fraction of the photoproduct K can be estimated as 0.52 ± 0.02. From the photo-steady-state concentrations of ppR and K and the calculated spectrum of K (A, curve 3), the quantum efficiency ratio of the forward and back light reaction, ppR  K, can be estimated (Balashov et al., 1991b) as 0.7 ± 0.1. This estimate is approximate because it does not take into account the heterogeneity of the bathoproduct K (Hirayama et al., 1992).

Besides ppR'₄₉₆, illumination of M at 160°C produced another photoproduct, which absorbed at shorter wavelengths. We term this photoproduct M' (or M'₄₀₄). It is characterized by bands at 380 and 404 nm that appear in the absorption spectrum after illumination of M at 160°C (see Fig. 3 A, curve 2). These sharp bands are well resolved in the second derivative spectrum (see maxima at 380, 404, and 417 nm in Fig. 5 A, which are absent in the spectrum of M in Fig. 3 C). The difference absorption spectrum accompanying formation of M' from M shows maxima at 402 and 378 nm due to the M' formation (Fig. 5 B) and minima at 390, 368, and 412 nm, which are close to the position of vibronic bands of initial M. The photoproduct M'₄₀₄ did not disappear after photoconversion of ppR'₄₉₆ back to M and thus is distinct from ppR'₄₉₆. Similar to M, M'₄₀₄ absorbs at short wavelengths and hence has an unprotonated Schiff base. More details on the spectrum of M'₄₀₄ were obtained upon thermal conversion of this intermediate (see below).

View larger version (17K): [in this window] [in a new window]   FIGURE 5   (A) Minus second derivative of the absorption spectrum of the sample containing M after its irradiation with 360-420-nm light at 160°C (of spectrum 2 in Fig. 3 A). The 404-nm and 380-nm bands belong to the photoproduct M'404 formed upon 360-420-nm irradiation of M at 160°C (these bands are absent in the spectrum of M (Fig. 1 C). (B) Difference spectrum M'404 minus M. The spectrum was obtained as a result of subtraction of the absorption changes caused by the transformation of M into ppR' (spectrum 2 in Fig. 3 B, taken with a minus and multiplied by a scaling factor, 1.29) from the difference spectrum caused by the transformation of M into both ppR' and M' (spectrum 1 in Fig. 3 B).

Thermal conversions of ppR'₄₉₆ and M'₄₀₄

Increasing the temperature of the sample containing ppR'₄₉₆ and M'₄₀₄, from 160°C to 110°C, and cooling back to 160°C resulted in an increase of absorbance and a redshift of the absorption maximum of the photoproduct from 496 to 504 nm (Fig. 6 A, curves 1 and 2). A subsequent increase in the temperature to 60°C and cooling back to 160°C caused a blueshift of the absorption maximum from 504 to 501 nm (Fig. 6 A, curve 3). The latter is peculiar to the initial ppR at 160°C. These data indicate that during thermal conversions of M' and ppR'₄₉₆ an intermediate is formed at 110°C, with the absorption maximum redshifted compared to initial ppR (to at least 504 nm). We will call this photoproduct ppR'₅₀₄. At 60°C all intermediates converted into the initial ppR (or to a spectroscopically identical species).

View larger version (17K): [in this window] [in a new window]   FIGURE 6   Thermal conversions of M'404 and ppR'496. (A) Absorption spectra of the photoproducts of M produced (1) by illumination of the sample containing M at 160°C with 360-420-nm light (same as spectrum 1 in Fig. 3 B); (2) after a subsequent increase in the temperature to 110°C and cooling back to 160°C; (3) after an increase in the temperature to 60°C and cooling back to 160°C. (B) Difference spectra caused by thermal transformations of M'404 and ppR'496. (1) Absorption changes observed in the sample containing M'404 and ppR'496 (sample 2 in Fig. 3 A) produced by increasing the temperature from 160°C to 60°C and cooling back to 160°C. (2) Same as 1, but most of ppR'496 was converted back to M (sample 3 in Fig. 3 A), so the changes are caused mostly by the thermal conversions of M'404. For the purpose of comparison of the spectrum of M'404 and initial M, the difference spectrum of M minus ppR'496 is given (curve 3). This is the same spectrum as in Fig. 3 B, curve 2, but multiplied by 0.25 to scale it with the other spectra.

The difference absorption changes accompanying thermal conversions of M' and ppR'₄₉₆ with an increase in the temperature from 160°C to 60°C are shown in Fig. 6 B, curve 1. The minima at 362, 382, 404, and 420 nm correspond to the vibronic bands of M'₄₀₄. Two bands, at 382 nm and 404 nm, have almost the same amplitude. The positive band with the maximum at 509 nm is composed of two difference spectra caused by two thermal transitions: M'₄₀₄  ppR and ppR'₄₉₆  ppR. The thermal transformation of M'₄₀₄ can be observed separately from the transformation of ppR'₄₉₆ (Fig. 6 B, curve 2) when most of ppR'₄₉₆ is photoconverted back to M at 160°C, as shown in Fig. 3 A, curve 3. As the sample containing M'₄₀₄ was warmed to 60°C, the M'₄₀₄ intermediate underwent transformation into a species with an absorption maximum at 503 nm, which was probably mostly initial ppR. A small redshift from 501 to 503 nm in this spectrum was most likely caused by a residual amount of ppR'₄₉₆ converting to ppR. As one can see from the difference spectra in Fig. 6 B (curves 1 and 2), the photoproduct M'₄₀₄ has a distinctive vibrational structure with the bands at 417, 404, 381, and 365 nm, which overlap but are clearly different from the bands of M at 414, 390, and 369 nm (Fig. 6 B, curve 3). The two long-wavelength bands of M'₄₀₄ are redshifted and narrower compared to the 414-nm and 390-nm bands of M.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Features of M photoconversion in ppR

The results presented above clearly indicate that the M intermediate of pharaonis phoborhodopsin (pharaonis sRII) is photoactive. The phototransformation of M involves formation of at least three intermediates, M'₄₀₄, ppR'₄₉₆, and ppR'₅₀₄. M'₄₀₄ presumably is the photoproduct of the primary light reaction of M, which most likely involves isomerization of the chromophore. The sharper vibronic bands of M'₄₀₄ indicate that the chromophore is more planar in M'₄₀₄ than in M, consistent with the 13-cis  all-trans isomerization. The photoreaction M  M' results in a dramatic increase in the proton affinity of the Schiff base because the long-wavelength species ppR'₄₉₆ with a protonated Schiff base appears even at 160°C, whereas reprotonation of the Schiff base during thermal decay of M occurs only above 40°C. In the M'  ppR' transition the proton is most likely taken from the counterion, Asp⁷⁵. The data indicate that phototransformation of M in ppR, like the phototransformation of M in bR (Litvin and Balashov, 1977; Balashov and Litvin, 1981b; Balashov, 1995), occurs through a pathway that is different from that used in the thermal transformation of M (Fig. 7). The simplest scheme of M photoconversion inferred from our data would be the following linear sequence:
However, we cannot exclude the possibility that the M intermediate and its primary photoproduct(s) M' are heterogeneous and that there are parallel pathways in the M photoconversion. Evidence for this is that at 160°C part of M is photoconverted into ppR'₄₉₆, whereas some fraction of the pigment stays in M'₄₀₄. This indicates the existence of two M' intermediates with different thermal stabilities: one is transformed into ppR'₄₉₆ at 160°C, and the other (M'₄₀₄) is stable at this temperature. To observe this less stable photoproduct, we have to examine the photoreaction of the M intermediate of ppR at lower temperatures. In bacteriorhodopsin, irradiation of M at 190°C does result in the formation of two primary photoproducts with different thermal stabilities (Balashov and Litvin, 1981b; Takei et al., 1992; Friedman et al., 1994).

View larger version (16K): [in this window] [in a new window]   FIGURE 7   A simplified scheme of the photochemical conversions of pharaonis phoborhodopsin (ppR, or psRII). Excitation of the pigment with blue-green (500 nm light) causes a cyclic reaction in which deprotonation of the Schiff base and formation of the M intermediate take place (Hirayama et al., 1992; Chizhov et al., 1998). Presumably M and the subsequent O state act as signaling states for the photophobic reaction of the cell (Yan et al., 1991). Formation and decay of the O intermediate is accompanied by proton uptake and release, correspondingly (Sasaki and Spudich, 1999; Iwamoto et al., 1999b). Photoexcitation of M with a near-UV light (360-420 nm) results in the photoconversion of M back to the initial state. This pathway involves formation of several intermediates found in this study, the primary photoproduct of M, M'404, and subsequent thermal products, ppR'496 and ppR'504, designated as M' and ppR' in the scheme. Thermal transformation from M' to ppR' is accompanied by reprotonation of the Schiff base, presumably from its counterion, Asp75, designated by letter A. The photoproduct ppR' is photoactive. Upon absorption of a light quantum it is transformed into M.

Because photoconversion of M into ppR'₄₉₆ occurs at 160°C, it is unlikely that the required proton for this transition is taken from the frozen bulk solution; the proton might be transported from the counterion (Asp⁷⁵), as in the case of bR (Balashov and Litvin, 1981b; Takei et al., 1992). The results suggest that photoconversion of M interrupts the photocycle of ppR and converts the pigment back to the initial state through a pathway that bypasses the N and O intermediates. Deprotonation of Asp⁷⁵ in the O  ppR transition is the rate-limiting step in the photocycle of ppR. Photoexcitation of M presumably induces proton transport from Asp⁷⁵ back to the Schiff base and subsequent reformation of the initial state. This hypothesis is in agreement with a recent study of Schmies et al. (2000), which showed that under certain conditions blue light illumination of pharaonis sRII (ppR) decreased the photocurrent generated by background green light illumination. These results indicate photoactivity of the M intermediate and phototransformation of M through a pathway different from its thermal conversion.

Photoproduct ppR'₄₉₆ is photoactive, and at 160°C it is transformed into M upon illumination. This is similar to the phototransformation of the photoproduct P565 formed at 160°C upon irradiation of the M intermediate of bacteriorhodopsin (Litvin and Balashov, 1977; Balashov and Litvin, 1981a). The redshifted photoproduct ppR'₅₀₄ may be analogous to the redshifted species P585, which is formed in the photoconversion of the M intermediate of bR at 60°C (Balashov and Litvin, 1981a; Balashov et al., 1988). The different species with a protonated Schiff base that are formed in the course of phototransformation of M into the initial state most likely represent certain steps in reformation of the Schiff base-counterion environment peculiar to the initial pigment. In bR they differ in hydrogen bonding of the protonated Schiff base and its counterion with internal water molecules that stabilize the ion pair (Maeda et al., manuscript submitted for publication). These water molecules participate in the formation of the M intermediate of bacteriorhodopsin (Luecke et al., 1999a). Internal water molecules might be involved in the photoreactions of ppR and its M intermediate as well and might participate in the formation of the ppR'₄₉₆ and ppR'₅₀₄ species, along with conformational changes of other important groups involved in the formation of M.

Implications of photoreversibility of the M intermediate on signal transduction in phoborhodopsin

In bacteriorhodopsin phototransformation of M prevents transmembrane proton transport. Based on the similarity of the photoreactions of the M intermediate in ppM and bR, we suggest that photoconversion of M into ppR might result in deactivation of the photoreceptor's signaling state. There is evidence that phototransduction of a light-induced signal occurs through the direct interaction of pigment with its phototransducer protein HtrII (Sasaki and Spudich, 1998). This interaction decreases the lifetime of the M and O intermediates. The latter indicates that the interaction with HtrII accelerates deprotonation of Asp⁷⁵, which has been slowed, perhaps because of the absence of a proton-releasing group. (In ppR the residue analogous to Glu¹⁹⁴ (Balashov et al., 1997; Dioumaev et al., 1998) is absent and the O intermediate has a very long lifetime.) If proton transfer from the Schiff base to Asp⁷⁵ and elimination of the Schiff base-counterion electrostatic interaction is a prerequisite for the generation of the ppR signaling state (Spudich et al., 1997), then the M  ppR' photoreaction should deactivate this signaling state by inducing fast reverse proton transport from the counterion back to the Schiff base. If signal generation is associated with the M or O decay, for instance, involving proton transport from the counterion to Htr during the O decay, then the photoreversal of M back to ppR will prevent this. One would expect that absorption of two quanta, a 500-nm quantum by ppR and a second 380-nm quantum by M, would result in no signal at all. The photoconversion of M would cause a decrease in the quantum yield of ppR similar to a decrease in the quantum yield of proton transport in bR (Ormos et al., 1978; Litvin et al., 1981). On the other hand, if signal generation is associated directly with the M formation (with the proton transport from the Schiff base to the counterion), then absorption of a quantum by M and photoconversion of M may result in a signal, opposite to the one generated upon excitation of initial ppR, as observed for sRI and its photoproduct M (Hoff et al., 1997). Further studies are needed to clarify the exact mechanism of coupling of the photocycle reactions with signal generation in ppR. Photoconversion of the M intermediate provides a useful tool for testing different scenarios.

Protein absorption changes during M formation and M photoconversion

Substantial light-induced changes of Trp environment and hydrogen bonding might be a part of a signal transduction mechanism, as has been specifically suggested for Trp¹²⁶ and Trp²⁶⁵ in rhodopsin to meta-II transition (Lin and Sakmar, 1996; Kochendoerfer et al., 1997). Several tryptophan and tyrosine residues constitute or are close to the chromophore-binding site in bacteriorhodopsin: Tyr⁵⁷, Tyr⁸³, Trp⁸⁶, Trp¹⁸², Tyr¹⁸⁵, Trp¹⁸⁹ (Grigorieff et al., 1996; Belrhali et al., 1999; Luecke et al., 1999b). Some of them undergo substantial conformational changes and changes in hydrogen bonding upon formation of M (Luecke et al., 1999a). These residues are conserved in ppR and other retinal proteins of halobacteria (Seidel et al., 1995; Shimono et al., 1998; Mukohata et al., 1999). Formation of the M intermediate in ppR is accompanied by an increase in absorption at 298 and 287 nm. The amplitude of absorption changes at 287 nm corresponds to changes in extinction of ~2200 cm¹/l mol (which is ~40% of the maximum extinction of a tryptophan residue). The 298-nm and 287-nm peaks can be caused by a small redshift of the absorption band of a tryptophan residue(s) and perhaps an increase in extinction due to a transition into a less polar environment or to changes in the electrostatic field (Balashov et al., 1991a; Wu et al., 1991) produced by a proton transport from the Schiff base to Asp⁷⁵. Similar (but more intensive) peaks at 297 and 288 nm are observed upon formation of the M intermediate in bacteriorhodopsin (Fig. 2 B and earlier spectra obtained under similar conditions; Sabés et al., 1984; Roepe et al., 1987). These peaks were attributed to perturbation of the environment of two to four tryptophan residues (Sabés et al., 1984). Studies of the time-resolved light-induced changes at 296 nm in the tryptophan mutants of bR led to identification of Trp¹⁸² as the residue responsible for the 296-nm peak in the M minus bR spectrum (Wu et al., 1991). The nature of the shallow minimum at 272 nm in the spectrum of M minus bR is not quite clear. Elimination of hyperchromic interactions is a possible cause for the decrease in absorbance around 280 nm (Lewis et al., 1997). It may also partially originate from a redshift of a Trp residue (Roepe et al., 1987). This minimum is absent in the M minus ppR difference spectrum. The origin of the differences in the light-induced changes in UV that accompanied formation of the M intermediates in ppR and bR needs further investigation. It may be caused by differences in aromatic residues (for example, Trp¹³⁷ in bR is replaced with Phe in ppR) or their involvement in the light-induced structural changes and proton transport. In bacteriorhodopsin the formation of the M intermediate not only includes proton transport from the Schiff base to the counterion but is also coupled to the conformational changes in the extracellular channel, leading to a proton release from a complex of residues, including Glu²⁰⁴, Glu¹⁹⁴, and water molecules (Balashov et al., 1997; Dioumaev et al., 1998). In ppR, a carboxylic residue analogous to Glu¹⁹⁴ is absent (is replaced with Pro¹⁸³), and proton release does not take place upon M formation but occurs during O decay (Sasaki and Spudich, 1999; Iwamoto et al., 1999b) and is accelerated in the presence of HtrII (Sasaki and Spudich, 1999). It is possible that these differences in coupling of the M intermediate with the proton transfer reactions in bR and ppR are relevant to the different perturbations of aromatic residues upon M formation in these two pigments.

In conclusion: We have found that the M intermediate of the photochemical cycle of pharaonis phoborhodopsin (pharaonis sensory rhodopsin II) is photoconvertible. The phototransformation of M involves several intermediate states and results in the fast light-induced reprotonation of the Schiff base and reformation of the initial pigment (or a spectrally isochromic species). Photoconversion of M comprises an additional pathway in halobacterial photoreception and may be used as a tool for the elucidation of the mechanism of signal transduction by phoborhodopsin.