CHEMICAL-REACTIVITY AND SPECTROSCOPY OF THE THIOL ESTER-LINKED P-COUMARIC ACID CHROMOPHORE IN THE PHOTOACTIVE YELLOW PROTEIN FROM ECTOTHIORHODOSPIRA-HALOPHILA

We have recently identifed p-coumaric acid as the chromophore of the p hotoactive yellow protein (PYP) from the purple sulfur bacterium Ecrot hiorhodospira halophiln, a blue-light photoreceptor with rhodopsin-lik e photochemistry [Hoff, W. D., Dux, P., Hard, K., Nugteren-Roodzant, I . M., Crielaard, W., Boelens, R., Kaptein, R., Van Beeumen, J., & Hell ingwerf, K. J. (1994) Biochemistry 33, 13959-13962]. Here we report on the chemistry of the linkage of this new photoactive cofactor to apoP YP: (i) Analysis of chromophore-peptide conjugates of PYP by high-reso lution mass spectrometry unambiguously shows that the p-coumaric acid molecule is bound to Cys 69 via a thiol ester bond. The PYP chromophor e is the first cofactor known to be stably thiol ester-linked to its a poprotein. (ii) The chemical reactivity of this thiol ester bond with respect to dithiothreitol, performic acid, and high pH is similar to t hat of disulfide bridges. These treatments result in the cleavage of t he thiol ester bond, concomitant with strong shifts in the UV/vis abso rbance band of the chromophore. (iii) The spectral properties of the P YP chromophore under different conditions are related to the structura l 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 ph otoreceptor proteins.