Infrared crystallography of the Oxygen Evolving Complex reactions of Photosystem II

了解氧气释放的分子过程是氧合光合作用研究中的一大挑战。结构信息是帮助和指导进展的基础，最近的突破做出了重大贡献，特别是从X射线结晶学和EXAFS研究。然而，最近的这些结构研究存在模棱两可的地方，部分原因是X射线辐射引起的还原，以及对偏振EXAFS测量的解释并不是唯一的。因此，需要一种新的结构方法来解决这些模棱两可的问题，并为五核Mn4Ca-簇合物的反应提供更多的洞察力，在那里发生水氧化和释放氧气。我们提出了PSII放氧复合体(OEC)的新测量方法，它克服了辐射问题，不仅提供了关于它的结构的信息，还提供了它的催化机理的信息。利用在衍射极限处聚焦的线偏振红外辐射，将测定从长链热球菌分离的PSII单晶的OEC中伴随S态跃迁的二色性红外吸收变化。这将在X射线框架中产生反应基团的振动跃迁偶极矩矢量，从而直接提供对Mn团簇及其配体的光诱导变化的结构测量。这项技术实际上相当于红外结晶学，它重要的是不会导致OEC中高价锰的辐射损伤或减少，作为一种结构技术，它将补充EXAFS和X射线结晶学来探索机理反应。迫切需要将这样一种新的结构技术应用于光合作用水氧化以及与之相关的S态转变这一重要问题。OEC的红外结晶学是一种结构技术，具有极大的附加值，可以极大地增强传统的非偏振FTIR差示光谱以及补充X射线衍射和X射线光谱分析的分析能力。通过使用现有的PSII晶体进行的初步实验，证明了这种测量的可行性。此外，我们已经证明，来自活性制剂的信号的幅度将大大超过所提议的仪器的检测极限。我们建议建立一个专门的实验室仪器来开发这项技术，并提供OEC功能的独特和重要的细节。此外，偶极梯度理论计算的发展建立在相关领域以前的工作的基础上，并将支持对单晶中红外二向色性的解释。在频率、截面和偶极子矢量的内部一致性与严格的理论建模相结合的一次实验中的实验可用性将提供对这一重要问题的结构和力学方面的洞察

Understanding the molecular process of oxygen evolution is one of the great challenges in oxygenic photosynthesis research. Structural information is fundamental to aid and guide progress where recent breakthroughs have contributed substantially, particularly from X-ray crystallographic and EXAFS studies. However ambiguities exist in these recent structural studies in part because of X-ray radiation induced reduction and because the interpretation of polarised EXAFS measurements are not unique. Therefore a new structural methodology is needed in order to address these ambiguities and to provide added insight in the reactions at the pentanuclear Mn4Ca-cluster complex where water oxidation occurs and dioxygen is released. We propose novel measurements of Oxygen Evolving Complex (OEC) of PSII which overcome radiation problems and will give information not only about its structure but also its catalytic mechanism. Using linearly polarised infrared radiation focussed at the diffraction limit, the dichroic infrared absorption changes that accompany the S-state transitions in the OEC of single crystals of PSII isolated from Thermosynechococcus elongatus will be determined. This will yield vibrational transition dipole moment vectors of reactive groups in the X-ray frame and thus directly provides structural measurement of light induced changes of the Mn cluster and its ligands. This technique in effect amounts to infrared crystallography, which importantly does not cause radiation induced damage or reduction of high valency Mn in the OEC and as a structural technique will complement EXAFS and X-ray crystallography to probe the mechanistic reactions. There is a pressing need for such a novel structural technique to be applied to the important problem of photosynthetic water oxidation and the associated S-state transitions. Infrared crystallography of the OEC is a structural technique that has the added value to greatly enhance the analytical power of conventional unpolarised FTIR difference spectroscopy as well as complement X-ray diffraction and X-ray spectroscopy. The feasibility of such measurements have been demonstrated through preliminary experiments using existing PSII crystals. Additionally we have shown that the amplitudes of the signals from the active preparations will be significantly in excess of the detection limit of the proposed instrumentation. We propose the setting-up of a dedicated laboratory based instrument to exploit the technique and provide unique and important details of OEC functioning. Furthermore, developments in theoretical calculation of dipole gradients build on previous work in related areas and will support the interpretation of infrared dichroism in single crystals. The experimental availability within one experiment with internal consistency of frequencies, cross sections and dipole vectors in combination with rigorous theoretical modelling will provide structural and mechanistic insight into this important problem