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

Hoff, Wouter D.; Devreese, Bart; Fokkens, Roel H.; Nugteren-Roodzant, I M; Beeumen, Jozef Van; Nibbering, N. M. M.; Hellingwerf, Klaas J.

doi:10.1021/bi951755z

Cited by 57 publications

(58 citation statements)

References 29 publications

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“…In agreement with this, it was observed that the position of the absorbance maximum of this presumed anionic form of the chromophore in pB, is tuned measurably from its position in aqueous solvents (i.e. 400 nm (34)) to ϳ425 nm. Hydrogen bonding between the phenolate moiety and Arg-52 (8) may contribute to this tuning of pB.…”

Section: Discussionsupporting

confidence: 62%

“…could even be effected by having the sample (making use of the slight pH drift) go through the pH transition range (ϳpH 7.8). Changes in the protonation level of PYP cannot be measured outside of the pH range from 4 to 11, because at a pH Ͻ 4.0 significant amounts of pB dark are formed (14), and at pH Ͼ 11 the thiol ester linkage in PYP starts to hydrolyze at a significant rate (34). The noise on the signal from the pH electrode increases slightly at alkaline pH, but it is always less than 0.05 H ϩ /PYP in the set-up used.…”

Section: Fig 3 Reversible (De)protonation Of Pyp At Acidic Neutralmentioning

confidence: 99%

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

Hendriks

Hoff²,

Crielaard

et al. 1999

Journal of Biological Chemistry

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Light-dependent pH changes were measured in unbuffered solutions of wild type photoactive yellow protein (PYP) and its H108F and E46Q variants, using two independent techniques: transient absorption changes of added pH indicator dyes and direct readings with a combination pH electrode. Depending on the absolute pH of the sample, a reversible protonation as well as a deprotonation can be observed upon formation of the transient, blue-shifted photocycle intermediate (pB) of this photoreceptor protein. The latter is observed at very alkaline pH, the former at acidic pH values. At neutral pH, however, the formation of the pB state is not paralleled by significant protonation/deprotonation of PYP, as expected for concomitant protonation of the chromophore and deprotonation of Glu-46 during pB formation. We interpret these results as further evidence that a proton is transferred from Glu-46 to the coumaric acid chromophore of PYP, during pB formation. One cannot exclude the possibility, however, that this transfer proceeds through the bulk aqueous phase. Simultaneously, an amino acid side chain(s) (e.g. His-108) changes from a buried to an exposed position. These results, therefore, further support the idea that PYP significantly unfolds in the pB state and resolve the controversy regarding proton transfer during the PYP photocycle.

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Section: Discussionsupporting

confidence: 62%

Section: Fig 3 Reversible (De)protonation Of Pyp At Acidic Neutralmentioning

confidence: 99%

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

Hendriks

Hoff²,

Crielaard

et al. 1999

Journal of Biological Chemistry

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“…A stronger hydrogen bonding lowers the energy barrier for proton transfer from Glu46 to pCA (Figure 4). In the pR to pB transition, Glu46 becomes deprotonated, concomitant with the protonation of pCA (Baca et al, 1994;Hoff et al, 1996). This fact, together with the strong hydrogen bonding between Glu46 and pCA in pR, strongly argues that a direct proton transfer occurs from the hydroxyl group of Glu46 to the phenolic oxygen of pCA.…”

Section: Resultsmentioning

confidence: 99%

Glu46 Donates a Proton to the 4-Hydroxycinnamate Anion Chromophore During the Photocycle of Photoactive Yellow Protein

et al. 1996

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Photoactive yellow protein (PYP) is a photoreceptor containing a unique 4-hydroxycinnamic acid (pCA) chromophore. The trans to cis photoisomerization of this chromophore activates a photocycle involving first a short-lived red-shifted intermediate (pR), then a long-lived blue-shifted intermediate (pB), and finally recovery of the original receptor state (pG). The pCA chromophore is deprotonated in pG and protonated in pB, but the proton donor for this process has not yet been identified. Here we report the first FTIR spectroscopic data on pG, pR, and pB. The IR difference signals in the carbonyl stretching region of COOH groups (1700−1800 cm-1) reveal that a buried carboxylic group close to the chromophore (i) is protonated in pG, (ii) develops a stronger hydrogen bonding in pR, and (iii) becomes deprotonated in pB. These signals are unambiguously assigned to Glu46, on the basis of the IR data and the 1.4 Å X-ray structure of PYP [Borgstahl et al. (1995) Biochemistry 34, 6278−6287]. Our data demonstrate that in pR Glu46 remains in hydrogen bonding contact with the negatively charged phenolic oxygen of pCA after chromophore photoisomerization. This strongly implies that the chromophore is isomerized to the 7-cis 9-s-trans conformation in pR, resulting from co-isomerization of both the C7C8 and C9C10 bonds. In the pR to pB transition, Glu46 becomes deprotonated, concomitant with chromophore protonation. Therefore, we conclude that Glu46 functions as the proton donor for the protonation of pCA during the PYP photocycle. We propose a molecular mechanism in which intramolecular proton transfer in PYP leads to global protein conformational changes involved in signal transduction.

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“…2 Free pCA in aqueous solvents and at neutral pH absorbs maximally at 284 nm (20); however, within the chromophorebinding pocket in the apoprotein, the absorption of the chromophore is strongly red-shifted (to 446 nm). Three contributions to this shift have been identified (6,7,19): (i) formation of the thiol ester bond between pCA and Cys-69, (ii) deprotonation of the chromophore (21), and (iii) specific protein-chromophore interactions.…”

Section: ϫ3mentioning

confidence: 99%

“…PYP has been implicated to function as the photosensor in this response, since its absorption spectrum matches its wavelength dependence (4). Although PYP resembles the sensory rhodopsins both functionally and photochemically, its chromophore is not retinal but a novel type of chromophore, 4-hydroxycinnamic acid (5,6), linked to Cys-69 via a thiol ester bond (7). Therefore, PYP represents a unique type of photoreceptor.…”

mentioning

confidence: 99%

Spectral Tuning, Fluorescence, and Photoactivity in Hybrids of Photoactive Yellow Protein, Reconstituted with Native or Modified Chromophores

Kroon¹,

Hoff²,

Fennema

et al. 1996

Journal of Biological Chemistry

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Photoactive yellow proteins (PYPs) constitute a new class of eubacterial photoreceptors, containing a deprotonated thiol ester-linked 4-hydroxycinnamic acid chromophore. Interactions with the protein dramatically change the (photo)chemical properties of this cofactor. Here we describe the reconstitution of apoPYP with anhydrides of various chromophore analogues. The resulting hybrid PYPs, their acid-denatured states, and corresponding model compounds were characterized with respect to their absorption spectrum, pK for chromophore deprotonation, fluorescence quantum yield, and Stokes shift. Three factors contributing to the tuning of the absorption of the hybrid PYPs were quantified: (i) thiol ester bond formation, (ii) chromophore deprotonation, and (iii) specific chromophore-protein interactions. Analogues lacking the 4-hydroxy substituent lack both contributions (chromophore deprotonation and specific chromophore-protein interactions), confirming the importance of this substituent in optical tuning of PYP. Hydroxy and methoxy substituents in the 3-and/or 5-position do not disrupt strong interactions with the protein but increase their pK for protonation and the fluorescence quantum yield. Both deprotonation and binding to apoPYP strongly decrease the Stokes shift of chromophore fluorescence. Therefore, coupling of the chromophore to the apoprotein not only reduces the energy gap between its ground and excited state but also the extent of reorganization between these two states. Two of the PYP hybrids show photoactivity comparable with native PYP, although with retarded recovery of the initial state.

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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

Cited by 57 publications

References 29 publications

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

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

Glu46 Donates a Proton to the 4-Hydroxycinnamate Anion Chromophore During the Photocycle of Photoactive Yellow Protein

Spectral Tuning, Fluorescence, and Photoactivity in Hybrids of Photoactive Yellow Protein, Reconstituted with Native or Modified Chromophores

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