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 unique 4-hydroxycinnamic acid (pCA) chromophore. The trans to cis photoisomerization of this chromophore activates a photocycle involving first a short-lived red-shifted intermediate (pR), then a long-lived blue-shifted intermediate (pB), and finally recovery of the original receptor state (pG). The pCA chromophore is deprotonated in pG and protonated in pB, but the proton donor for this process has not yet been identified. Here we report the first FTIR spectroscopic data on pG, pR, and pB. The IR difference signals in the carbonyl stretching region of COOH groups (1700-1800 cm-1) reveal that a buried carboxylic group close to the chromophore (i) is protonated in pG, (ii) develops a stronger hydrogen bonding in pR, and (iii) becomes deprotonated in pB. These signals are unambiguously assigned to Glu46, on the basis of the IR data and the 1.4 A X-ray structure of PYP [Borgstahl et al. (1995) Biochemistry 34, 6278-6287]. Our data demonstrate that in pR Glu46 remains in hydrogen bonding contact with the negatively charged phenolic oxygen of pCA after chromophore photoisomerization. This strongly implies that the chromophore is isomerized to the 7-cis 9-s-trans conformation in pR, resulting from co-isomerization of both the C7 = C8 and C9-C10 bonds. In the pR to pB transition, Glu46 becomes deprotonated, concomitant with chromophore protonation. Therefore, we conclude that Glu46 functions as the proton donor for the protonation of pCA during the PYP photocycle. We propose a molecular mechanism in which intramolecular proton transfer in PYP leads to global protein conformational changes involved in signal transduction.
Xie
A
A
Department of Physiology and Biophysics, Albert Einstein College of Medicine, Bronx, New York 10461, USA.
Hoff
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WD
Kroon
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AR
Hellingwerf
K J
KJ
D001426:Bacterial Proteins / Q000737:chemistry*; D004926:Escherichia coli; D018698:Glutamic Acid / Q000737:chemistry*; D006090:Gram-Negative Bacteria / Q000235:genetics; D006860:Hydrogen Bonding; D010777:Photochemistry; D020283:Photoreceptors, Microbial; D011522:Protons; D011994:Recombinant Proteins; D017550:Spectroscopy, Fourier Transform Infrared
D001426:Bacterial Proteins; D020283:Photoreceptors, Microbial; D011522:Protons; D011994:Recombinant Proteins; C050162:photoactive yellow protein, Bacteria; D018698:Glutamic Acid