The photocycle intermediates of photoactive yellow protein (PYP) were chara
cterized by low-temperature Fourier transform infrared spectroscopy, The di
fference FTIR spectra of PYPB, PYPH, PYPL, and PYPM minus PYP were measured
under the irradiation condition determined by UV-visible spectroscopy. Alt
hough the chromophore bands Of PYPB were weak, intense sharp bands compleme
ntary to the 1163-cm(-1) band of PYP. which show the chromophore is deproto
nated, were observed at 1168-1169 cm(-1) for PYPH and PYPL, indicating that
the proton at Glu46 is not transferred before formation of PYPM. Free tran
s-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 b
ands 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 wer
e shifted by deuterium-labeling at the ethylene bond of the chromophore whi
le labeling at the phenol part had no effect, they were attributed to the e
thylene bond region. Hence, structural differences among the intermediates
are present in this region. Bands at about 1730 cm(-1), which show that Glu
46 is protonated, were observed for all intermediates except for PYPM. Beca
use the frequency of this mode was constant in PYPB, PYPH, and PYPL, the en
vironment of Glu46 is conserved in these intermediates. The photocycle of P
YP would therefore proceed by changing the structure of the twisted ethylen
e bond of the chromophore.