Zhang H, Razeghifard M R, Fischer G, Wydrzynski T
Research School of Biological Sciences, Institute of Advanced Studies, The Australian National University, Canberra, Australia 0200.
Biochemistry. 1997 Sep 30;36(39):11762-8. doi: 10.1021/bi970815t.
In this paper, we present the first time-dependent measurements of flash-induced infrared difference spectra of photosystem II (PSII) using Fourier transform infrared (FTIR) spectroscopy. With this experimental approach, we were able to obtain the YZoxQA-/YZQA vibrational difference spectrum of Tris-washed, PSII-enriched samples in the absence of hydroxylamine at room temperature (16 +/- 2 degrees C), with a spectral resolution of 4 cm-1 and a temporal resolution of 50 ms. In order to determine the dominant species in the FTIR spectrum at a particular point in time after an excitation flash, the decay kinetics of YZox and QA- were independently monitored by EPR and chlorophyll a fluorescence, respectively, under the same experimental conditions. These measurements confirmed that the addition of DCMU to Tris-washed PSII samples does not significantly affect the YZox decay, but does substantially slow down the QA- decay. By making use of the difference in the decay kinetics using DCMU, the QA-/QA signals could be separated from the YZox/YZ signals and a pure QA-/QA difference spectrum obtained. By comparison of the YZoxQA-/YZQA difference spectrum with the pure QA-/QA difference spectrum, a large differential band at 1706/1699 cm-1 could be identified and associated with YZ oxidation. In contrast, an intense band at 1478 cm-1, whose DCMU-sensitive decay follows the QA- decay based on the chlorophyll a fluorescence measurements, was present in all of the time-resolved spectra. Since no significant reversible Chl+ radicals could be detected by the EPR measurements under our experimental conditions, we confirm that this band most likely arises only from the semiquinone anion QA- [Berthomieu, C., Nabedryk, E., Mäntele, W., & Breton, J. (1990) FEBS Lett. 269, 363-367].
在本文中，我们首次使用傅里叶变换红外（FTIR）光谱对光系统II（PSII）的闪光诱导红外差光谱进行了时间分辨测量。通过这种实验方法，我们能够在室温（16±2℃）下，在不存在羟胺的情况下，获得经Tris洗涤、富含PSII的样品的YZoxQA-/YZQA振动差光谱，光谱分辨率为4 cm-1，时间分辨率为50 ms。为了确定激发闪光后特定时间点FTIR光谱中的主要物种，在相同实验条件下，分别通过电子顺磁共振（EPR）和叶绿素a荧光独立监测YZox和QA-的衰减动力学。这些测量证实，向经Tris洗涤的PSII样品中添加敌草隆（DCMU）不会显著影响YZox的衰减，但会大幅减缓QA-的衰减。利用使用DCMU时衰减动力学的差异，可以将QA-/QA信号与YZox/YZ信号分离，并获得纯QA-/QA差光谱。通过将YZoxQA-/YZQA差光谱与纯QA-/QA差光谱进行比较，可以识别出在1706/1699 cm-1处的一个大的差异带，并将其与YZ氧化相关联。相比之下，在所有时间分辨光谱中都存在一个位于1478 cm-1处的强带，基于叶绿素a荧光测量，其对DCMU敏感的衰减遵循QA-的衰减。由于在我们的实验条件下通过EPR测量未检测到明显的可逆叶绿素+自由基，我们证实该带很可能仅来自半醌阴离子QA- [贝托米厄，C.，纳贝德里克，E.，曼特勒，W.，& 布雷顿，J.（1990）《欧洲生物化学学会联合会快报》269，363 - 367]。
