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

Abstract

The unique ability of photoactive proteins to capture and use energy from a photon of light depends on the chromophore, its linkage to the protein, and the surrounding protein environment. To understand the molecular mechanisms by which a chromophore and protein interact to undergo a light cycle, we are studying photoactive yellow protein (PYP), a 14-kDa water-soluble photoreceptor from Ectothiorhodospira halophila with a photocycle similar to that of sensory rhodopsin. Here, we report the cloning and sequencing of the pyp gene and the chemical identification of both the chromophore and its covalent linkage to the protein. Elemental composition data from high-resolution mass spectrometry of a proteolytically derived chromopeptide, pH titrations and UV-visible spectroscopy of the protein-bound and chemically released chromophore, and fragmentation mass spectrometry of the liberated chromophore amide were combined with results from the 1.4-A-resolution protein crystal structure to identify the chromophore in PYP as a 4-hydroxycinnamyl group covalently bound to the sole cysteine residue via a thioester linkage. While 4-hydroxycinnamate is a metabolic product of the phenylpropanoid pathway and a key molecule in plant stress response, this is the first report of covalent modification of a protein by this group. In the dark (yellow) state of PYP, the protein stabilizes the chromophore as the deprotonated phenolate anion. By combining our biochemical characterization of the chromophore with other published observations, we propose a chemical basis for the photocycle: following the initial absorption of a photon, the photocycle of PYP involves protonation of the chromophore to a neutral phenol form corresponding to the observed photobleached intermediate.

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

“…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.…”

“…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).…”

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

“…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.…”

“…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.…”

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

“…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.…”