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

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 PYP M , 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 PYP M . A 1736 cm ؊1 band was observed for PYP, but the corresponding band for PYP M was not. Because it disappeared in the Glu-46 3 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 PYP M .Photoactive yellow protein (PYP) 1 ( max ϭ 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 ␣/␤ 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 Ϫ190°C yields PYP B ( max ϭ 489 nm) and PYP H ( max ϭ 442 nm), which are thermally converted to PYP L ( max ϭ 456 nm) through PYP BL ( max ϭ 400 nm) and PYP HL ( max ϭ 447 nm), respectively. The two pathways beginning with PYP B and PYP H join at PYP L and revert to PYP. Flash photolysis at ambient temperature identified two intermediates, pR ( max ϭ 465 nm) and pB ( max ϭ 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 PYP L (10) (In this paper, pR and pB are called PYP L and PYP M , respectively, to avoid confusion) and that PYP L is accumulated by irradiation of PYP at Ϫ80°C (10, 13). However, the precursors of PYP L have not been discovered by flash photolysis at room temperature.Recent studies have clarified some details of the events that take place during t...

Protonation/Deprotonation Reactions Triggered by Photoactivation of Photoactive Yellow Protein from Ectothiorhodospira halophila

“…In addition, it was proposed that during pB formation, proton transfer occurs from Glu-46 to the chromophore. Subsequent extension of these Fourier transform infrared analyses, using isotope enrichment and variants of PYP obtained through site-directed mutagenesis, provided further evidence supporting this proposal (22). However, timeresolved x-ray crystallography of PYP in the nanosecond time domain indicates that at room temperature the hydrogen bond between the side chain of Glu-46 and the phenolic oxygen of the chromophore is already disrupted in the pR intermediate (25).…”

“…The two most extreme views are that this proton transfer may take place directly, within the chromophore binding pocket inside the protein, or indirectly, through the bulk solvent. As the chromophore of PYP during pB formation is protonated (8) and Glu-46 is deprotonated (18,22), net proton uptake by the protein while it progresses through the photocycle is difficult to understand. Therefore, we have investigated these reversible (de)protonation reactions, using not only transient absorption spectroscopy, in combination with pH indicator dyes, but also transient pH measurements with a sensitive combination pH electrode.…”

“…In the E46Q mutant of PYP (13,22), direct protonation of the chromophore by its hydrogen-bonding partner in the chromophore-binding pocket cannot take place. It may therefore be anticipated that in this derivative, upon formation of pB a proton has to be taken up from the bulk aqueous phase.…”

On the absorbance changes in the photocycle of the photoactive yellow protein: A quantum-chemical analysis

“…During the bleaching of the protein an arginine gateway opens, allowing solvent exposure and protonation of the phenolic oxygen to occur. Nevertheless, that the amino acid residue Glu-46 acts as the actual proton donor to the chromophore has been seen with IR techniques (32,33). The cycle is completed when the PYP G structure is recovered by means of a protein-mediated thermal process (34).…”

Initial Events in the Photocycle of Photoactive Yellow Protein

“…The trans-4-hydroxy cinnamic acid chromophore (HC4) is present as a phenolate anion in a hydrophobic pocket in the protein, where it is covalently attached to the S␥ of Cys 69 by a thioester bond (3,4). In the dark state of PYP (denoted pG; max ϭ 446 nm), the phenolic oxygen (O-4Ј) of the chromophore is stabilized by hydrogen bonds to Tyr 42 and to the protonated carboxyl group of Glu 46 (5)(6)(7). During the photocycle the ethylene double bond in the chromophore (C-2ϭC-3) isomerizes from the trans-configuration to the cis-configuration (8).…”