Noguchi T, Tomo T, Kato C
Biophysical Chemistry Laboratory, RIKEN, Wako, Saitama 351-0198, Japan.
Biochemistry. 2001 Feb 20;40(7):2176-85. doi: 10.1021/bi0019848.
The process of formation of the triplet state of chlorophyll in the photosystem II (PS II) reaction center complex was studied by means of time-resolved infrared (IR) spectroscopy. Using a dispersive-type IR spectrometer with a time resolution of approximately 55 ns, transient spectra in the C=O stretching region (1760--1600 cm(-1)) were measured at 77 K. The data were analyzed by singular-value decomposition and subsequent least-squares fitting. Two distinct spectral components having different kinetic behaviors were resolved. One had spectral features characterized by negative peaks at 1740 and 1680 cm(-1) and an overall positive background and was assigned to the P(680)(+)Phe(-)/P(680)Phe radical pair by static FTIR measurements of the P(680)(+)/P(680) and Phe(-)/Phe differences. The other had prominent negative and positive peaks at 1668 and 1628 cm(-1), respectively, which were previously assigned to the keto C==O change upon triplet formation of the monomeric chlorophyll denoted as Chl(T) [Noguchi, T., Tomo, T., and Inoue, Y. (1998) Biochemistry 37, 13614-13625]. The former component of P(680)(+)Phe(-)/P(680)Phe exhibited a multiphasic decay with time constants of 77 ns (75%), 640 ns (18%), 8.3 micros (4%), and 0.3 ms (3%), while the latter component of (3)Chl(T)/Chl(T) was formed with a single-exponential rise with a time constant of 57 ns and had a lifetime of 1.5 ms. From the observations that only the two spectral components were resolved without any other triplet intermediates and the time constant of (3)Chl(T) formation roughly agreed with or seemed even faster than that of the major phase of the P(680)(+)Phe(-) decay, two alternative mechanisms of triplet formation are proposed. (i) (3)Chl(T) is directly formed from P(680)(+)Phe(-) by charge recombination at Chl(T), and (ii) (3)P(680) is formed, and then the triplet is transferred to Chl(T) with a time constant of much less than 50 ns. The location of Chl(T) in the D1 subunit as the monomer chlorophyll corresponding to the accessory bacteriochlorophyll in the L subunit of purple bacteria is favored to explain the former mechanism as well as the triplet properties reported in the literature. The physiological role of the triplet formation on Chl(T) is also discussed.
利用时间分辨红外（IR）光谱法研究了光系统II（PS II）反应中心复合物中叶绿素三重态的形成过程。使用时间分辨率约为55 ns的色散型红外光谱仪，在77 K下测量了C=O伸缩区域（1760 - 1600 cm⁻¹）的瞬态光谱。通过奇异值分解和随后的最小二乘法拟合对数据进行分析。分辨出了具有不同动力学行为的两个不同光谱成分。一个具有在1740和1680 cm⁻¹处有负峰以及整体正背景的光谱特征，通过对P(680)⁺/P(680)和Phe⁻/Phe差异的静态傅里叶变换红外（FTIR）测量，将其归属于P(680)⁺Phe⁻/P(680)Phe自由基对。另一个在1668和1628 cm⁻¹处分别有突出的负峰和正峰，先前已将其归属于单体叶绿素三重态形成时酮C==O的变化，记为Chl(T) [野口智、友磨彻、井上洋（1998年）《生物化学》37卷，13614 - 13625页]。P(680)⁺Phe⁻/P(680)Phe的前一个成分表现出多相衰减，时间常数分别为77 ns（75%）、640 ns（18%）、8.3 μs（4%）和0.3 ms（3%），而(³)Chl(T)/Chl(T)的后一个成分以57 ns的时间常数单指数上升形成，寿命为1.5 ms。基于仅分辨出这两个光谱成分而无任何其他三重态中间体以及(³)Chl(T)形成的时间常数大致与P(680)⁺Phe⁻衰减的主要相的时间常数相符甚至似乎更快的观察结果, 提出了两种三重态形成的替代机制。(i) (³)Chl(T)通过在Chl(T)处的电荷复合直接由P(680)⁺Phe⁻形成，以及(ii) 形成(³)P(680)，然后三重态以远小于50 ns的时间常数转移至Chl(T)。Chl(T)在D1亚基中的位置作为与紫色细菌L亚基中的辅助细菌叶绿素相对应的单体叶绿素，有利于解释前一种机制以及文献中报道的三重态性质。还讨论了Chl(T)上三重态形成的生理作用。
