Imamoto Y, Shirahige Y, Tokunaga F, Kinoshita T, Yoshihara K, Kataoka M
Graduate School of Materials Science, Nara Institute of Science and Technology, Ikoma, Nara 630-0101, Japan.
Biochemistry. 2001 Jul 31;40(30):8997-9004. doi: 10.1021/bi010021l.
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.
通过低温傅里叶变换红外光谱对光活性黄色蛋白（PYP）的光循环中间体进行了表征。在紫外可见光谱确定的辐照条件下，测量了PYP（B）、PYP（H）、PYP（L）和PYP（M）减去PYP的差示傅里叶变换红外光谱。尽管PYP（B）的发色团谱带较弱，但在1168 - 1169 cm⁻¹处观察到与PYP的1163 cm⁻¹谱带互补的强尖锐谱带，表明发色团去质子化，这在PYP（H）和PYP（L）中出现，表明在PYP（M）形成之前，Glu46处的质子未转移。游离反式对香豆酸有一个1294 cm⁻¹的谱带，其顺式形式移至1288 cm⁻¹。所获得的所有差示傅里叶变换红外光谱都有与之对应的一对谱带，表明所有中间体的发色团均处于顺式构型。1020 - 960 cm⁻¹处的特征振动模式区分了这些中间体。由于这些模式在发色团的乙烯键处进行氘标记时发生了位移，而在酚部分进行标记时没有影响，因此它们归因于乙烯键区域。因此，中间体之间的结构差异存在于该区域。除了PYP（M）之外，在所有中间体中都观察到了约1730 cm⁻¹处的谱带，表明Glu46是质子化的。因为该模式的频率在PYP（B）、PYP（H）和PYP（L）中是恒定的，所以在这些中间体中Glu46的环境是保守的。因此，PYP的光循环将通过改变发色团扭曲乙烯键的结构来进行。
