COMMUNICATION:
Evidence for Proton Transfer from Glu-46 to the Chromophore during the Photocycle of Photoactive Yellow Protein*

(Received for publication, January 28, 1997, and in revised form, March 17, 1997)

Yasushi Imamoto Dagger , Ken'ichi Mihara Dagger , Osamu Hisatomi Dagger , Mikio Kataoka Dagger , Fumio Tokunaga Dagger §, Nina Bojkova and Kazuo Yoshihara

From the Dagger  Department of Earth and Space Science, Graduate School of Science, Osaka University, Toyonaka, Osaka 560, Japan and the  Suntory Institute for Bioorganic Research, 1-1, Wakayamadai, Shimamoto-cho, Osaka 618, Japan

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES


ABSTRACT

Photoactive yellow protein (PYP) belongs to the novel group of eubacterial photoreceptor proteins. To fully understand its light signal transduction mechanisms, elucidation of the intramolecular pathway of the internal proton is indispensable because it closely correlates with the changes in the hydrogen-bonding network, which is likely to induce the conformational changes. For this purpose, the vibrational modes of PYP and its photoproduct were studied by Fourier transform infrared spectroscopy at -40 °C. The vibrational modes characteristic for the anionic p-coumaryl chromophore (Kim, M., Mathies, R. A., Hoff, W. D., and Hellingwerf, K. J. (1995) Biochemistry 34, 12669-12672) were observed at 1482, 1437, and 1163 cm-1 for PYP. However, the bands corresponding to these modes were not observed for PYPM, the blue-shifted intermediate, but the 1175 cm-1 band characteristic of the neutral p-coumaryl chromophore was observed, indicating that the phenolic oxygen of the chromophore is protonated in PYPM. A 1736 cm-1 band was observed for PYP, but the corresponding band for PYPM was not. Because it disappeared in the Glu-46 right-arrow Gln mutant of PYP, this band was assigned to the C=O stretching mode of the COOH group of Glu-46. These results strongly suggest that the proton at Glu-46 is transferred to the chromophore during the photoconversion from PYP to PYPM.


INTRODUCTION

Photoactive yellow protein (PYP)1 (lambda max = 446 nm) (1) is proposed to be a photoreceptor protein for the negative phototaxis observed in the purple phototrophic bacterium, Ectothiorhodospira halophila (2). PYP belongs to the novel group of photoreceptor proteins (3, 4) whose structures are quite different from those of the other photoreceptor proteins studied so far. Namely, the protein moiety of PYP has an alpha /beta fold structure (5) composed of 125 amino acids (6, 7). The chromophore is a p-coumaric acid (7-9) bound to a cysteine residue via a thioester bond.

PYP absorbs a photon and enters the photocycle. We have analyzed the photocycle of PYP in detail by low temperature spectroscopy and identified several intermediates (10). Irradiation of PYP at -190 °C yields PYPB (lambda max = 489 nm) and PYPH (lambda max = 442 nm), which are thermally converted to PYPL (lambda max = 456 nm) through PYPBL (lambda max = 400 nm) and PYPHL (lambda max = 447 nm), respectively. The two pathways beginning with PYPB and PYPH join at PYPL and revert to PYP. Flash photolysis at ambient temperature identified two intermediates, pR (lambda max = 465 nm) and pB (lambda max = 355 nm) (11, 12). pR is formed within 10 ns after flash excitation. It decays to pB over a submillisecond time scale and reverts to PYP within 1 s. It has been demonstrated that pR is the same species as PYPL (10) (In this paper, pR and pB are called PYPL and PYPM, respectively, to avoid confusion) and that PYPL is accumulated by irradiation of PYP at -80 °C (10, 13). However, the precursors of PYPL have not been discovered by flash photolysis at room temperature.

Recent studies have clarified some details of the events that take place during the PYP photocycle. Crystallography at 1.4-Å resolution (7) and resonance Raman spectroscopy (14) have shown that the phenolic oxygen of the chromophore of PYP is deprotonated in the dark state. The photocycle is initiated by photon absorption, which involves isomerization of the ethylenic bond of the chromophore (15). During the photocycle, observed proton uptake and release correspond with the formation and decay of PYPM (16).

The largely blue-shifted absorption spectrum of PYPM suggests that the chromophore/protein interaction of PYPM is quite different from that of PYP. Therefore, elucidation of the chromophore structure of PYPM is essential to understand the photocycle of PYP. For the spectral blue-shift of PYPM, the following explanations would be possible: (i) the phenolic oxygen of the chromophore is protonated; (ii) the conjugated double bond system is broken by extreme distortion of the chromophore (25); and (iii) the pi  electrons of the conjugated double bond system are localized by a nearby positive charge (e.g. Arg-52) (26). The first explanation is the most simple and plausible, but there has been no previous experimental evidence to support it, and the others could not be excluded.

In the dark state, the phenolic oxygen of the chromophore interacts with the OH groups of Tyr-42 and Glu-46 by hydrogen bonds (5). As shown in retinal protein systems, the changes in the hydrogen-bonding network centering on the chromophore closely correlates with the protein conformational changes (17). Therefore, it is of importance to study the internal proton movement around the chromophore and nearby amino acid residues to understand the light signal transduction mechanism of PYP. Recently it has been reported that the C=O stretching mode of the COOH group disappears on the formation of PYPM (18). They reasoned that Glu-46 donates a proton to the chromophore, based on the fact that Glu-46 has a unique COOH group embedded in the protein. This idea arose on elucidation of the tertiary structure of PYP (5) and seems reasonable. However, two vital pieces of experimental evidence required to support this conclusion have never been reported: one is that the chromophore of PYPM is protonated, and the other is assignment of the C=O stretching mode.

Recent progress in experimental techniques made it possible to prepare PYP with an isotope-labeled chromophore (9) and a site-directed mutant of PYP (19, 27). These techniques have enabled assignment of the vibrational modes. In the present study, to study the movement of the proton around the chromophore and nearby amino acid residues, the vibrational modes of the chromophore and COOH group of PYP and its intermediates were analyzed by low temperature FTIR spectroscopy. The first experimental findings indicating proton transfer from Glu-46 to the chromophore during the photocycle are presented.


MATERIALS AND METHODS

PYP was isolated from E. halophila BN 9626 according to previously reported methods (1, 10). The Glu-46 right-arrow Gln mutant of PYP (E46Q) was expressed by Escherichia coli and reconstituted with p-coumaric anhydride (19). p-Coumaric-8-13C-acid was prepared by p-hydroxybenzaldehyde and triethylphosphonoacetate-2-13C followed by alkaline hydrolysis of the ester. 13C-Labeled PYP was prepared by reconstitution of PYP with 13C-labeled p-coumaric anhydride and apoPYP (9). They were then desalted by dialysis and applied to a small DEAE-Sepharose column (Pharmacia Biotech Inc.). After washing the column with 10 mM phosphate buffer (pH 7.2), PYP was eluted with a linear gradient of NaCl (100-200 mM) in the same buffer. PYP was then concentrated with an ultrafiltration membrane (Centriprep 10, Amicon) and diluted with 10 mM phosphate buffer. Dilution and concentration steps were repeated several times to remove NaCl. Finally, PYP was concentrated to 3-4 mg/ml.

A 20 µl sample was placed on a BaF2 window (10 mm in diameter) and dried under a gentle stream of N2 gas. The dried sample was sealed using a silicon rubber spacer and another BaF2 window and set in the sample cell holder. Before sealing, 0.2 µl of H2O or D2O was put inside the spacer for hydration or deuteration of PYP. The sample cell holder was mounted in an optical cryostat (DN1704, Oxford) connected to a temperature controller (ITC502, Oxford).

Absorption spectra in the UV-visible region were recorded with a Hitachi U-3210 recording spectrophotometer. Infrared spectra were recorded with a Horiba FT-210 Fourier transform infrared spectrophotometer equipped with an MCT detector. The sample was irradiated with a 1 kW slide projector (HILUX-HR, Tokyo Master) using glass optical filters (Y47, Toshiba). The difference FTIR spectra shown in this paper are the means of four to eight independent recordings, each of which was the mean of 64 scans (resolution = 2 cm-1).


RESULTS

Our recent low temperature spectroscopy results with PYP in 66% glycerol buffer showed that PYPL is formed by the irradiation of PYP at -80 °C, but PYPM was not observed in the thermal reaction (10). This could be due to the effect of the presence of glycerol at high concentration, because it suppresses the decay of PYPL but accelerates the decay of PYPM (20). In the method used here, the film sample can be frozen with no increase in turbidity, and the addition of glycerol was not necessary for the low temperature experiments, thus removing impediments to the formation of PYPM. We tested several irradiation conditions above -80 °C to accumulate PYPM without PYPL for FTIR spectroscopy. Upon irradiation of PYP film with >450-nm light at -40 °C, the absorbance at 350 nm was increased (Fig. 1). Because this product had largely blue-shifted absorption spectrum similar to pB (Fig. 1, inset) and directly reverted to PYP (data not shown), it would correspond to pB (called PYPM hereafter). In this difference spectrum, the absorbance at 460 nm was not increased, indicating that only PYPM was formed. Under this irradiation condition, the difference FTIR spectra were recorded with hydrated and deuterated PYP films. The difference spectra before and after irradiation with >450-nm light at -40 °C are the difference between PYPM and PYP (PYPM/PYP spectrum), in which positive and negative peaks are attributed to PYPM and PYP, respectively (Fig. 2). The prominent bands of PYP were observed at 1736, 1560, 1482, 1437, 1301, 1163, 1058, 1041, 983, and 831 cm-1. On the other hand, the intensities of the bands of PYPM were relatively small, but 1175, 1081, and 994 cm-1 bands were characteristic for PYPM. Several bands of them were affected by D2O substitution.


Fig. 1. UV-visible spectroscopy on hydrated PYP film. The dried PYP hydrated with 0.2 µl of H2O was cooled to -40 °C (curve 1) and irradiated with >450-nm light for 5, 10, 20, 40, 80, 160, 320, or 640 s at -40 °C (curves 2-9, respectively). Inset, the difference spectra before and after the irradiation were calculated by subtracting curve 1 from curves 2-9.
[View Larger Version of this Image (24K GIF file)]


Fig. 2. Difference FTIR spectra between PYP and PYPM in hydrated (solid line) and deuterated (dotted line) samples. The IR spectrum of each sample was recorded before and after irradiation with >450-nm light for 600 s (means of 64 scans at -40 °C). After warming the sample to the room temperature, recording was repeated. The spectra shown were normalized at 1163 cm-1.
[View Larger Version of this Image (27K GIF file)]

Recent resonance Raman spectroscopy covering the range 1750-1000 cm-1 (14) was helpful for the implication of our FTIR data. It was reported that the prominent vibrational modes of PYP were observed at 1633, 1558, 1498, 1439, 1288, 1165, 1059, and 1043 cm-1 in H2O buffer (14) and were shifted 0-4 cm-1 by D2O substitution. It was concluded that the chromophore of PYP is deprotonated; otherwise the chromophore bands would be D2O-sensitive. It was further pointed out that the deprotonated chromophore shows bands at 1498, 1439 and 1165 cm-1 but when protonated there is only one band at 1176 cm-1 (14). The PYPM/PYP spectrum reported here shows corresponding bands: negative at 1482, 1437, and 1163 cm-1 and positive at 1175 cm-1. The bands at 1482, 1437, and 1163 cm-1 were observed only on the negative side, indicating that PYPM does not have vibrational modes corresponding to these. Moreover, the fact that the 1175 cm-1 band was strongly affected by D2O substitution suggests that the chromophore of PYPM is protonated. To confirm that the 1175 cm-1 band of PYPM is the vibrational mode of the chromophore, PYPM/PYP spectrum was recorded using PYP containing 13C-labeled chromophore at 8 position under the same irradiation condition (>450-nm light, -40 °C) (Fig. 3a). The shift of 1175 cm-1 band with 13C-label (Fig. 3a) was similar to that with D2O substitution (Fig. 2). Therefore, this band is attributed to the chromophore, which proves that the chromophore of PYPM is protonated.


Fig. 3. Assignments of the vibrational modes. a, 1300-1000 cm-1 region of PYPM/PYP spectra of 13C-labeled PYP (solid line) and unlabeled PYP (dotted line). b, 1800-1650 cm-1 region of the PYPM/PYP spectra of wild type PYP (dotted line) and E46Q (solid line). The spectra were normalized at the 1163 cm-1 band.
[View Larger Version of this Image (28K GIF file)]

The negative band at 1736 cm-1 should be noted because it could be the C=O stretching mode of the COOH group of either aspartic acid or glutamic acid. A similar band was found recently by FTIR spectroscopy (18), although the irradiation condition was considerably different from that of the present experiment. The appearance of the negative band without the complementary positive band indicates deprotonation of the COOH group. Among 19 acidic residues of PYP (7), the most plausible candidate for the origin of this band would be Glu-46 because its OH group directly interacts with the phenolic oxygen of the chromophore and it is buried inside the protein. To test this, a Glu-46 right-arrow Gln mutant (E46Q) of PYP was prepared (19), and its vibrational modes were studied by FTIR spectroscopy.

Whereas the absorption maximum of E46Q in the visible region is slightly red-shifted from wild type PYP (19), the intermediate corresponding to PYPM accumulated under the same irradiation conditions (>450-nm light, -40 °C) (data not shown). FTIR spectroscopy was then carried out using E46Q, and a PYPM/PYP spectrum was obtained (Fig. 3b). As expected, the PYPM/PYP spectrum of E46Q did not show a 1736-cm-1 band, indicating that the C=O stretching mode of the COOH group of Glu-46 is indeed the origin of this band. Namely, Glu-46 is protonated in PYP and releases its proton in the PYPM state.


DISCUSSION

Based on our present observations, the model for proton movement during the PYP photocycle is shown in Fig. 4. On absorption of a photon, the chromophore of PYP isomerizes to the cis form (15). There are two possible models for the isomerization of the chromophore: one is that the phenol part rotates, and the other is that the ester part rotates. Xie et al. (18) proposed that the hydrogen bond between the OH group of Glu-46 and the phenolic oxygen of the chromophore remains in PYPL and supported the latter model. However, they irradiated PYP at 80 K to trap PYPL. At 80 K, irradiation yields a mixture of PYPB and PYPH (10, 21), so the changes in C=O stretching mode of PYP observed at 80 K cannot be attributed to PYPL. Moreover, the intermediate formed readily absorbs a photon, and it is uncertain whether or not the photoproduct accumulated at 80 K has the cis chromophore. Therefore, few data are available to discuss the mechanism of the isomerization of the chromophore. However, the latter model is likely because the phenolic oxygen interacts with Glu-46 and Tyr-42, and it is fixed by the hydrogen bond. In the case of retinal proteins, the protein moiety has the binding site for the beta -ionone ring of the chromophore, and the Schiff base portion (rather than the beta -ionone ring region) rotates on photoisomerization (22). That is, in both PYP and retinal protein systems, the smaller moiety rotates on isomerization. Minimization of the conformational change upon isomerization would contribute to the fast and highly efficient photoreaction of photoreceptor proteins.


Fig. 4. Schematic drawing of proton movement in PYP. In the dark, the phenolic oxygen of the chromophore is deprotonated and Glu-46 is protonated. On photoconversion from PYP to PYPM, the proton is transferred from Glu-46 to the chromophore. The chromophore of PYP is in the trans form (7, 15) whereas that of PYPM is in the cis form (15).
[View Larger Version of this Image (18K GIF file)]

The present experiments have demonstrated the first evidence that the phenolic oxygen of the chromophore is protonated in PYPM. The absorption maximum of PYPM is 355 nm (12), which is close to that of denatured PYP at neutral pH (11, 23). Because the phenolic oxygen of PYP is protonated, the largely blue-shifted absorption spectrum of PYPM is due mainly to protonation of the chromophore. However, the possibility that steric interactions and the nearby positive charge affect the absorption spectrum of PYPM is not completely excluded. These effects will be examined in future research.

In retinal proteins, the chromophore of the dark state is protonated at the nitrogen atom of the Schiff base linkage and positively charged. It is deprotonated and neutral in the near-UV intermediates like M and metarhodopsin II. In contrast, the p-coumaryl chromophore is negatively charged in PYP but is neutral in PYPM. Despite the striking differences in the chromophores, protein moieties, and linkages, the chromophores of both retinal proteins and PYP turn neutral in their near-UV intermediates, which are thought to be formed as the result of large conformational changes (17, 28). In addition, cis/trans isomerization of the chromophore is involved in both systems (15). These are the common events for the protein conformational changes among the photoreceptor proteins and would be the most efficient mechanism. Namely, the charged chromophore is the core of the hydrogen-bonding network that determines its tertiary structure in the dark state. Isomerization of the chromophore on photon absorption results in the loss of its charge. As a result, the hydrogen-bonding network is disordered, and the protein conformational change takes place. In PYP, the phenolic oxygen of the chromophore interacts with Tyr-42 and Glu-46, and the chromophore binding site is Cys-69. The loss of this interaction would make the structure of the chromophore binding site remarkably flexible.

The present work confirmed that the 1736 cm-1 band of PYP is the C=O stretching mode of the COOH group of Glu-46. Absence of this mode in PYPM indicates that Glu-46 is protonated in PYP but deprotonated in PYPM. Concurrently, the chromophore of PYPM is protonated. This indicates that the pKa values of the chromophore and Glu-46 of PYPM are close to those in solution but that those of PYP are considerably different due to the hydrophobic environment in the protein. Therefore, the chromophore binding pocket would be exposed to the solvent in PYPM as the result of the protein conformational changes.

Because the protonation states of the chromophore and Glu-46 are complementary, the simplest model for proton pathways would be that the proton at Glu-46 is transferred to the chromophore in PYPM state, recovering to Glu-46 in the recovery step (Fig. 4). In E46Q, the chromophore would uptake proton from the solvent or Tyr-42 would act as the alternative proton donor. This model does not explain the proton release and uptake coupled with the formation and decay of PYPM. Therefore, another amino acid residue and/or water might be involved in the proton transfer, resulting in vectorial movement of the proton, as with bacteriorhodopsin (24). Further FTIR studies on the other intermediates using various site-directed mutants will elucidate the pathway of proton transfer.


FOOTNOTES

*   This work was supported in part by a grants-in-aid from the Japanese Ministry of Education, Culture, and Science.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§   To whom correspondence should be addressed: Dept. of Earth and Space Science, Graduate School of Science, Osaka University, Toyonaka, Osaka 560, Japan. Tel.: 81-6-850-5499; Fax: 81-6-850-5480; E-mail: tokunaga{at}ess.sci.osaka-u.ac.jp.
1   The abbreviations used are: PYP, photoactive yellow protein from E. halophila; FTIR, Fourier transform infrared.

ACKNOWLEDGEMENTS

We thank Dr. Keizo Shimada of Tokyo Metropolitan University for the kind gift of E. halophila strain BN 9626. We also thank Dr. Ian Gleadall for comments on the manuscript and Yuji Shirahige for technical assistance.


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