COMPLETE CHEMICAL-STRUCTURE OF PHOTOACTIVE YELLOW PROTEIN - NOVEL THIOESTER-LINKED 4-HYDROXYCINNAMYL CHROMOPHORE AND PHOTOCYCLE CHEMIST

The unique ability of photoactive proteins to capture and use energy f rom a photon of light depends on the chromophore, its linkage to the p rotein, and the surrounding protein environment. To understand the mol ecular mechanisms by which a chromophore and protein interact to under go a light cycle, we are studying photoactive yellow protein (PYP), a 14-kDa water-soluble photoreceptor from Ectothiorhodospira halophila w ith a photocycle similar to that of sensory rhodopsin. Here, we report the cloning and sequencing of the pyp gene and the chemical identific ation 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 W-visible s pectroscopy 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-Angstrom-resolution protein c rystal structure to identify the chromophore in PYP as a 4-hydroxycinn amyl group covalently bound to the sole cysteine residue via a thioest er linkage. While 4-hydroxycinnamate is a metabolic product of the phe nylpropanoid pathway and a key molecule in plant stress response, this is the first report of covalent modification of a protein by this gro up. In the dark (yellow) state of PYP, the protein stabilizes the chro mophore as the deprotonated phenolate anion. By combining our biochemi cal characterization of the chromophore with other published observati ons, we propose a chemical basis for the photocycle: following the ini tial absorption of a photon, the photocycle of PYP involves protonatio n of the chromophore to a neutral phenol form corresponding to the obs erved photobleached intermediate.