Low-temperature Fourier transform infrared spectroscopy of photoactive yellow protein.
The photocycle intermediates of photoactive yellow protein (PYP) were characterized by low-temperature Fourier transform infrared spectroscopy. The difference FTIR spectra of PYP(B), PYP(H), PYP(L), and PYP(M) minus PYP were measured under the irradiation condition determined by UV-visible spectroscopy. Although the chromophore bands of PYP(B) were weak, intense sharp bands complementary to the 1163-cm(-1) band of PYP, which show the chromophore is deprotonated, were observed at 1168-1169 cm(-1) for PYP(H) and PYP(L), indicating that the proton at Glu46 is not transferred before formation of PYP(M). Free trans-p-coumaric acid had a 1294-cm(-1) band, which was shifted to 1288 cm(-1) in the cis form. All the difference FTIR spectra obtained had the pair of bands corresponding to them, indicating that all the intermediates have the chromophore in the cis configuration. The characteristic vibrational modes at 1020-960 cm(-1) distinguished the intermediates. Because these modes were shifted by deuterium-labeling at the ethylene bond of the chromophore while labeling at the phenol part had no effect, they were attributed to the ethylene bond region. Hence, structural differences among the intermediates are present in this region. Bands at about 1730 cm(-1), which show that Glu46 is protonated, were observed for all intermediates except for PYP(M). Because the frequency of this mode was constant in PYP(B), PYP(H), and PYP(L), the environment of Glu46 is conserved in these intermediates. The photocycle of PYP would therefore proceed by changing the structure of the twisted ethylene bond of the chromophore.
Imamoto
Y
Y
Graduate School of Materials Science, Nara Institute of Science and Technology, Ikoma, Nara 630-0101, Japan. imamato@ms.aist-nara.ac.jp
Shirahige
Y
Y
Tokunaga
F
F
Kinoshita
T
T
Yoshihara
K
K
Kataoka
M
M
D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000528:radiation effects; D003373:Coumaric Acids / Q000737:chemistry; D003903:Deuterium; D005615:Freezing; D018698:Glutamic Acid / Q000235:genetics; D005973:Glutamine / Q000235:genetics; D020607:Halorhodospira halophila; D008027:Light; D010777:Photochemistry; D020283:Photoreceptors, Microbial; D011422:Propionates; D013056:Spectrophotometry, Ultraviolet; D017550:Spectroscopy, Fourier Transform Infrared / Q000379:methods
D001426:Bacterial Proteins; D003373:Coumaric Acids; D020283:Photoreceptors, Microbial; D011422:Propionates; C050162:photoactive yellow protein, Bacteria; D005973:Glutamine; D018698:Glutamic Acid; D003903:Deuterium; C495469:p-coumaric acid