Department of Physics and Astronomy, Faculty of Sciences, Vrije Universiteit, De Boelelaan 1081, 1081 HV Amsterdam, The Netherlands.
J Phys Chem B. 2010 Apr 1;114(12):4335-44. doi: 10.1021/jp9089326.
Protochlorophyllide (PChlide) is a precursor in the biosynthesis of chlorophyll. Complexed with NADPH to the enzyme protochlorophyllide oxidoreductase (POR), it is reduced to chlorophyllide, a process that occurs via a set of spectroscopically distinct intermediate states and is initiated from the excited state of PChlide. To obtain a better understanding of these catalytic events, we characterized the excited state dynamics of PChlide in the solvents tetrahydrofuran (THF), methanol, and Tris/Triton buffer using ultrafast transient absorption in the visible and mid-infrared spectral regions and time-resolved fluorescence emission experiments. For comparison, we present time-resolved transient absorption measurements of chlorophyll a in THF. From the combined analysis of these experiments, we derive that during the 2-3 ns excited state lifetime an extensive multiphasic quenching of the emission occurs due to solvation of the excited state, which is in agreement with the previously proposed internal charge transfer (ICT) character of the S1 state ( Zhao , G. J. ; Han , K. L. Biophys. J. 2008 , 94 , 38 ). The solvation process in methanol occurs in conjunction with a strengthening of a hydrogen bond to the Pchlide keto carbonyl group. We demonstrate that the internal conversion from the S2 to S1 excited states is remarkably slow and stretches out on to the 700 fs time scale, causing a rise of blue-shifted signals in the transient absorption and a gain of emission in the time-resolved fluorescence. A triplet state is populated on the nanosecond time scale with a maximal yield of approximately 23%. The consequences of these observations for the catalytic pathway and the role of the triplet and ICT state in activation of the enzyme are discussed.
原叶绿素（PChlide）是叶绿素生物合成的前体。与 NADPH 结合到酶原叶绿素氧化还原酶（POR）中，它被还原为叶绿素，这一过程通过一组光谱上不同的中间状态发生，并且是从 PChlide 的激发态开始的。为了更好地理解这些催化事件，我们使用超快瞬态吸收在可见和中红外光谱区域以及时间分辨荧光发射实验来表征四氢呋喃（THF）、甲醇和 Tris/Triton 缓冲液中 PChlide 的激发态动力学。为了比较，我们展示了 THF 中叶绿素 a 的时间分辨瞬态吸收测量。通过对这些实验的综合分析，我们得出结论，在 2-3 ns 的激发态寿命期间，由于激发态的溶剂化，发射会发生广泛的多相猝灭，这与之前提出的 S1 态的内部电荷转移（ICT）特性一致（Zhao，G. J.；Han，K. L. Biophys. J. 2008，94，38）。甲醇中的溶剂化过程伴随着与 Pchlide 酮羰基的氢键加强。我们证明，从 S2 到 S1 激发态的内转换非常缓慢，并延伸到 700 fs 的时间尺度，导致瞬态吸收中出现蓝移信号的上升和时间分辨荧光中发射的增加。三重态在纳秒时间尺度上被填充，最大产率约为 23%。这些观察结果对催化途径以及三重态和 ICT 态在酶激活中的作用的影响进行了讨论。
