GLU46 DONATES A PROTON TO THE 4-HYDROXYCINNAMATE ANION CHROMOPHORE DURING THE PHOTOCYCLE OF PHOTOACTIVE YELLOW PROTEIN

Photoactive yellow protein (PYP) is a photoreceptor containing a uniqu e 4-hydroxycinnamic acid (pCA) chromophore. The trans to cis photoisom erization of this chromophore activates a photocycle involving first a short-lived red-shifted intermediate (pR), then a long-lived blue-shi fted intermediate (pB), and finally recovery of the original receptor state (pG). The pCA chromophore is deprotonated in pG and protonated i n pB, but the proton donor for this process has not yet been identifie d. Here we report the first FTIR spectroscopic data on pc, pR, and pB. The IR difference signals in the carbonyl stretching region of COOH g roups (1700-1800 cm(-1)) reveal that a buried carboxylic group close t o the chromophore (i) is protonated in pG, (ii) develops a stronger hy drogen bonding in pR, and (iii) becomes deprotonated in pB. These sign als are unambiguously assigned to Glu46, on the basis of the IR data a nd the 1.4 Angstrom X-ray structure of PYP [Borgstahl et al. (1995) Bi ochemistry 34, 6278-6287]. Our data demonstrate that in pR Glu46 remai ns in hydrogen bonding contact with the negatively charged phenolic ox ygen of pCA after chromophore photoisomerization. This strongly implie s that the chromophore is isomerized to the 7-cis 9-s-trans conformati on in pR, resulting from co-isomerization of both the C-7=C-8 and C-9- C-10 bands. In the pR to pB transition, Glu46 becomes deprotonated, co ncomitant with chromophore protonation, Therefore, we conclude that Gl u46 functions as the proton donor for the protonation of PCA during th e PYP photocycle. We propose a molecular mechanism in which intramolec ular proton transfer in PYP leads to global protein conformational cha nges involved in signal transduction.