Infrared Spectroscopic Studies of the Photosynthetic Oxygen-Evolving Complex

技术上的光合作用放氧发生在植物、绿藻和蓝藻中。这一过程对于维持地球上的有氧、异养生命是必不可少的。本研究利用红外光谱和差示红外光谱技术，对光系统II的结构和功能进行了新的研究。该酶在光驱动下进行水的氧化成氧和叶绿体对苯二酚的还原反应。这个过程发生在一个含锰的催化位置，它可以积累从水中产生氧气所需的四个氧化当量。催化中心的顺序氧化形式被称为Sn态，其中n表示形成的氧化当量的数量。目前尚不清楚锰的结合部位、S态锰团簇的结构以及水氧化的化学机理。一种称为锰稳定蛋白的外在亚基，通过一种未知的机制改变了酶的催化性质。本研究的具体目的是：1.利用振动光谱继续研究S_1~S_2跃迁，验证S_1~S_2跃迁中Mn的氧化对与金属簇连接或接近的羧酸盐和羧酸残基的振动光谱产生扰动的假说；2.利用振动光谱将研究扩展到其他S态的跃迁，验证可以记录其他S态跃迁的差值红外光谱的假说，以获得有关光合作用水氧化机理的新信息；以及III.通过红外和其他光谱研究Mn团簇的光组装，验证了可以形成并利用红外和其他光谱研究Mn团簇光驱动组装的中间体的假设。这种中间体的形成可以用来获得更多关于催化部位的结构和功能的信息。这项研究的重点是光合作用中的放氧机制。进行光合作用放氧的蛋白质或酶存在于绿色植物、藻类和蓝藻或蓝藻中。这种产生氧气的光合作用是地球上人类生命的基础，因为这个过程会产生必要的营养物质，并保持地球大气中的氧气。从光获得的能量被用来驱动放氧的化学反应。除非提供能量输入，否则这些反应是不可能的。虽然光诱导放氧或水分解是一种普遍存在的非常重要的生物现象，但在这个过程中发生的化学反应对酶的了解很少。这项研究的重点是使用振动光谱来获得关于这些化学反应的更多信息。振动光谱学是一种测量原子振动频率和幅度的技术。这些频率和幅度反映了分子结构。通过使用振动光谱跟踪水解酶进行反应，将获得更多关于化学机理的信息。通过使用同位素标记和蛋白质诱变来解释光谱。这些实验有望为这一无处不在的重要生物过程提供新的信息。

98-08934Barry 1.Technical Photosynthetic oxygen evolution occurs in plants, green algae, and cyanobacteria. This process is essential for maintenance of aerobic, heterotrophic life on earth. In this study, the techniques of infrared and difference infrared spectroscopy are used to obtain new information about the structure and function of photosystem II. This enzyme carries out the light driven oxidation of water to form oxygen and reduction of plastoquinone to form plastoquinol. This process occurs at a Mn-containing catalytic site, which can accumulate the four oxidizing equivalents necessary to generate oxygen from water. The sequentially oxidized forms of the catalytic site are called the Sn states, where n refers to the number of oxidizing equivalents formed. The binding sites for Mn, the structures of the Mn cluster in the S states, and the chemical mechanism of water oxidation are not understood. An extrinsic subunit, called the Mn stabilizing protein, alters the catalytic properties of the enzyme by an as yet unknown mechanism. The specific aims of this study are: I. to continue studies of the S1 to S2 transition through the use of vibrational spectroscopy, testing the hypothesis that oxidation of Mn in the S1 to S2 transition perturbs the vibrational spectrum of carboxylate and carboxylic acid residues that are ligating to or close to the metal cluster; II. to expand studies to other S state transitions through the use of vibrational spectroscopy, testingthe hypothesis that the difference infrared spectra of other S state transitions can be recorded and used to obtain new information about the mechanism of photosynthetic water oxidation; and III. to study the photoassembly of the Mn cluster through the use of infrared and other spectroscopies, tesing the hypothesis that an intermediate in the light-driven assembly of the Mn cluster can be formed and studied using infrared and other spectroscopies. The formation of this intermediate can be used to obtain more information about the structure and function of the catalytic site.2. Non-technicalThis research is focused on the mechanism of oxygen evolution in photosynthesis. The protein or enzyme that carries out photosynthetic oxygen evolution is found in green plants, algae, and blue-green algae or cyanobacteria. This type of oxygen-producing photosynthesis is the basis of human life on earth, because the process produces essential nutrients and maintains oxygen in the earth's atmosphere. The energy derived from light is used to drive the chemical reactions of oxygen evolution. These reactions are not possible unless that energy input is supplied. Although light-induced oxygen evolution or "water-splitting" is a ubiquitous, very important biological phenomenon, the chemical reactions, which occur during this process on the enzyme, are poorly understood. The focus of this study is the use of vibrational spectroscopy to obtain more information about these chemical reactions. Vibrational spectroscopy is a technique in which the frequencies and amplitudes of atomic vibrations are measured. These frequencies and amplitudes reflect the structure of a molecule. By using vibrational spectroscopy to follow the water-splitting enzyme as the reactions are performed, more information about the chemical mechanism will be obtained. The spectra are interpreted through the use of isotopic labeling and protein mutagenesis. These experiments are expected to yield new information about this ubiquitous, important biological process.