Volume 272, Number 20, Issue of May 16, 1997 pp. 12905-12908
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.

COMMUNICATION:
Evidence for Proton Transfer from Glu-46 to the Chromophore during the Photocycle of Photoactive Yellow Protein

(Received for publication, January 28, 1997, and in revised form, March 17, 1997)

Yasushi Imamoto , Ken'ichi Mihara , Osamu Hisatomi , Mikio Kataoka , Fumio Tokunaga , Nina Bojkova ^¶ and Kazuo Yoshihara ^¶

From the  Department of Earth and Space Science, Graduate School of Science, Osaka University, Toyonaka, Osaka 560, Japan and the ^¶ Suntory Institute for Bioorganic Research, 1-1, Wakayamadai, Shimamoto-cho, Osaka 618, Japan

Photoactive yellow protein (PYP) belongs to the novel group of eubacterial photoreceptor proteins. To fully understand its light signal transduction mechanisms, elucidation of the intramolecular pathway of the internal proton is indispensable because it closely correlates with the changes in the hydrogen-bonding network, which is likely to induce the conformational changes. For this purpose, the vibrational modes of PYP and its photoproduct were studied by Fourier transform infrared spectroscopy at 40 °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 observed at 1482, 1437, and 1163 cm¹ for PYP. However, the bands corresponding to these modes were not observed for PYP_M, the blue-shifted intermediate, but the 1175 cm¹ band characteristic of the neutral p-coumaryl chromophore was observed, indicating that the phenolic oxygen of the chromophore is protonated in PYP_M. A 1736 cm¹ band was observed for PYP, but the corresponding band for PYP_M was not. Because it disappeared in the Glu-46  Gln mutant of PYP, this band was assigned to the C=O stretching mode of the COOH group of Glu-46. These results strongly suggest that the proton at Glu-46 is transferred to the chromophore during the photoconversion from PYP to PYP_M.

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