Complete Chemical Structure of Photoactive Yellow Protein: Novel Thioester-Linked 4-Hydroxycinnamyl Chromophore and Photocycle Chemistry

Baca, Manuel; Borgstahl, Gloria E. O.; Boissinot, Maurice; Burke, Patrick; Williams, Dewight; Slater, Kelly; Getzoff, Elizabeth D.

doi:10.1021/bi00252a001

Cited by 301 publications

(369 citation statements)

References 16 publications

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“…On this basis we initially proposed that the PYP chromophore is bound to the apoprotein via a disulfide bridge. However, subsequent experiments indicated that it is more likely that the chromophore is bound to PYP via a thiol ester (Hoff et al, 1994a;Baca et al, 1994). This point a The calculated exact masses for key ions generated upon positive ion FABMS from the heptapeptide-chromophore conjugate (Figures 2 and 3) are shown together with their measured masses and elemental composition.…”

Section: Resultsmentioning

confidence: 98%

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Chemical Reactivity and Spectroscopy of the Thiol Ester-Linked p-Coumaric Acid Chromophore in the Photoactive Yellow Protein from Ectothiorhodospira halophila

et al. 1996

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Chemical reactivity and spectroscopy of the thiol ester-linked p-coumaric acid chromophore in the photoactive yellow protein from Ectothiorhodospira halophila Hoff, W.D.; Devreese, B.; Fokkens, R.H.; Nugteren-Roodzant, J.M.; Beeumen, J.; Nibbering, N.M.M.; Hellingwerf, K.J. Published in: Biochemistry DOI:10.1021/bi951755zLink to publication Citation for published version (APA):Hoff, W. D., Devreese, B., Fokkens, R. H., Nugteren-Roodzant, J. M., Beeumen, J., Nibbering, N. M. M., & Hellingwerf, K. J. (1996). Chemical reactivity and spectroscopy of the thiol ester-linked p-coumaric acid chromophore in the photoactive yellow protein from Ectothiorhodospira halophila. Biochemistry, 35, 1274-1281/1284. DOI: 10.1021/bi951755z General rightsIt is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), other than for strictly personal, individual use, unless the work is under an open content license (like Creative Commons). Disclaimer/Complaints regulationsIf you believe that digital publication of certain material infringes any of your rights or (privacy) interests, please let the Library know, stating your reasons. In case of a legitimate complaint, the Library will make the material inaccessible and/or remove it from the website. Please Ask the Library: http://uba.uva.nl/en/contact, or a letter to: Library of the University of Amsterdam, Secretariat, Singel 425, 1012 WP Amsterdam, The Netherlands. You will be contacted as soon as possible. Here we report on the chemistry of the linkage of this new photoactive cofactor to apoPYP: (i) Analysis of chromophore-peptide conjugates of PYP by high-resolution mass spectrometry unambiguously shows that the p-coumaric acid molecule is bound to Cys 69 via a thiol ester bond. The PYP chromophore is the first cofactor known to be stably thiol ester-linked to its apoprotein.(ii) The chemical reactivity of this thiol ester bond with respect to dithiothreitol, performic acid, and high pH is similar to that of disulfide bridges. These treatments result in the cleavage of the thiol ester bond, concomitant with strong shifts in the UV/vis absorbance band of the chromophore. (iii) The spectral properties of the PYP chromophore under different conditions are related to the structural integrity of the protein, the presence of the thiol ester bond, and the ionization state of the phenolic proton of the chromophore. These results are important for the general problem of spectral tuning in photoreceptor proteins.

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Section: Resultsmentioning

confidence: 98%

“…However, the exact physical basis of this red shift, i.e., of the spectral tuning of chromophore absorbance by proteins in general, is not well understood. In the case of PYP the protonation state of the phenolic hydroxyl group of the chromophore has been proposed to be important (Baca et al, 1994).…”

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Chemical Reactivity and Spectroscopy of the Thiol Ester-Linked p-Coumaric Acid Chromophore in the Photoactive Yellow Protein from Ectothiorhodospira halophila

et al. 1996

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“…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)(8)(9) bound to a cysteine residue via a thioester bond.…”

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confidence: 99%

“…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)(8)(9) bound to a cysteine residue via a thioester bond.…”

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confidence: 99%

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

Imamoto

Mihara

Hisatomi

et al. 1997

Journal of Biological Chemistry

125

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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...

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“…As an example, the Racker band observed in the NAD + -bound form of E. coli glyceraldehyde-3-phosphate dehydrogenase results from a charge-transfer interaction between Cys159 and the nicotinamide ring (17,18). Many other possible structures could result in an absorption band at 315 nm, including various thioether/thioester cross-links like those found in photoactive yellow protein (19), apo-galactose oxidase (20), and model complexes of apo-galactose oxidase (21). However, because the chromophoric species is destroyed upon alkylation of selenophosphate synthetase, any cysteine cross-link must be reversible such that the nucleophilic thiol(ate) is accessible.…”

Section: Discussionmentioning

confidence: 99%

Mechanistic Insights Revealed Through Characterization of a Novel Chromophore in Selenophosphate Synthetase from Escherichia coli

Wolfe

2003

IUBMB Life

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SummaryThe incorporation of selenium into specific proteins and tRNAs requires selenophosphate (SePO 3 ), whose formation is catalyzed by selenophosphate synthetase. In a Mg/ATP-dependent reaction, selenophosphate synthetase catalyzes the phosphorylation of selenide to yield AMP, inorganic phosphate, and SePO 3 . In this report, a previously unrecognized chromophore covalently attached to selenophosphate synthetase is characterized. The UV/Vis spectrum of selenophosphate synthetase has a feature centered at 315 nm that is irreversibly destroyed by alkylation. Moreover, addition of Zn 2 + , which is known to inhibit selenophosphate synthetase, reversibly quenches the 315 nm absorption. Since Zn 2 + is known to bind to Cys17, these data strongly suggest that this residue participates in the 315 nm absorption. Upon incubation with both Mg 2 + and ATP, the l max of the chromophore shifts to 340 nm, and it is shown that the shift requires binding of nucleotide having a hydrolyzable g-phosphoryl group. These data indicate that either the chromophore is directly involved in phosphoryl transfer or indirectly reflects a phosphorylation-dependent conformational change in selenophosphate synthetase. This work provides the first spectroscopic handle on catalytic steps associated with SePO 3 synthesis, which will be used to study the molecular structure of the chromophore and its role in the catalytic mechanism of selenophosphate synthetase. IUBMB Life, 55: 689-693, 2003

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Complete Chemical Structure of Photoactive Yellow Protein: Novel Thioester-Linked 4-Hydroxycinnamyl Chromophore and Photocycle Chemistry

Cited by 301 publications

References 16 publications

Chemical Reactivity and Spectroscopy of the Thiol Ester-Linked p-Coumaric Acid Chromophore in the Photoactive Yellow Protein from Ectothiorhodospira halophila

Chemical Reactivity and Spectroscopy of the Thiol Ester-Linked p-Coumaric Acid Chromophore in the Photoactive Yellow Protein from Ectothiorhodospira halophila

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

Mechanistic Insights Revealed Through Characterization of a Novel Chromophore in Selenophosphate Synthetase from Escherichia coli

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