TRANS CIS (Z/E) PHOTOISOMERIZATION OF THE CHROMOPHORE OF PHOTOACTIVE YELLOW PROTEIN IS NOT A PREREQUISITE FOR THE INITIATION OF THE PHOTOCYCLE OF THIS PHOTORECEPTOR PROTEIN/

The chromophore of photoactive yellow protein (PYP) (i.e,, 4-hydroxyci nnamic acid) has been replaced by an analogue with a triple bond, rath er than a double bond (by using 4-hydroxyphenylpropiolic acid in the r econstitution, yielding hybrid I) and by a ''locked'' chromophore (thr ough reconstitution with 7-hydroxycoumarin-3-carboxylic acid, in which a covalent bridge is present across the vinyl bond, resulting in hybr id II). These hybrids absorb maximally at 464 and 413 nm, respectively , which indicates that in both hybrids the deprotonated chromophore do es fit into the chromophore-binding pocket. Because the triple bond ca nnot undergo cis/trans (or E/Z) photoisomerization and because of the presence of the lock across the vinyl double bond in hybrid II, it was predicted that these two hybrids would not be able to photocycle. Sur prisingly, both are able. We have demonstrated this ability by making use of transient absorption, low-temperature absorption, and Fourier-t ransform infrared (FTIR) spectroscopy. Both hybrids, upon photoexcitat ion, display authentic photocycle signals in terms of a red-shifted in termediate; hybrid I, in addition, goes through a blueshifted-like int ermediate state, with very slow kinetics.We interpret these results as further evidence that rotation of the carbonyl group of the thioester -linked chromophore of PYP, proposed in a previous FTIR study and visu alized in recent time-resolved x-ray diffraction experiments, is of cr itical importance for photoactivation of PYP.