EVIDENCE FOR PROTON-TRANSFER FROM GLU-46 TO THE CHROMOPHORE DURING THE PHOTOCYCLE OF PHOTOACTIVE YELLOW PROTEIN

Photoactive yellow protein (PYP) belongs to the novel group of eubacte rial photoreceptor proteins, To fully understand its light signal tran sduction mechanisms, elucidation of the intramolecular pathway of the internal proton is indispensable because it closely correlates with th e changes in the hydrogen-bonding network, which is likely to induce t he conformational changes, For this purpose, the vibrational modes of PYP and its photoproduct mere studied by Fourier transform infrared sp ectroscopy at -40 degrees C, The vibrational modes characteristic for the anionic p-coumaryl chromophore (Kim, M., Mathies, R. A., Hoff, W. D., and Hellingwerf, K. J. (1995) Biochemistry 34, 12669-12672) were o bserved at 1482, 1437, and 1163 cm(-1) for PYP, However, the bands cor responding to these modes were not observed for PYPM, the blue-shifted intermediate, but the 1175 cm(-1) band characteristic of the neutral p-coumaryl chromophore was observed, indicating that the phenolic oxyg en of the chromophore is protonated in PYPM, A 1736 cm(-1) band was ob served for PYP, but the corresponding band for PYPM was not, Because i t disappeared in the Glu-46 --> Gln mutant of PYP, this band was assig ned to the C=O stretching mode of the COOH group of Glu-46, These resu lts strongly suggest that the proton at Glu-46 is transferred to the c hromophore during the photoconversion from PYP to PYPM.