SPECTROELECTROCHEMISTRY OF FLAVO- AND METALLOPROTEINS

我们感兴趣的是确定蛋白质环境如何影响 氧化还原活性中心的性质；我们选择研究两类 含有两种中心的蛋白质，黄素蛋白和双核 铁蛋白，两者都能催化电子转移反应。我们的 目标是将氧化还原电位与蛋白质功能联系起来，以及 确定基板/组件如何进一步调整此功能 有约束力的。最终，我们试图确定特定的结构 控制酶功能和调节的活性部位的特征。 这两类蛋白质的成员具有共同的氧化还原特性：1) 它们有参与催化的氧化还原活性中心；2)它们是 能够进行一个或两个电子转移，利用唯一的自由基 混合价中间体；3)伴随电子转移的质子化 在热力学调节中很重要；以及4)它们的氧化还原性质 似乎受底物、产物或 调节蛋白成分。两组成员的结构 要么是X射线结晶学已知的，要么是正在进行深入研究的 其他光谱方法。我们提出的电化学数据 积累对于这两个班级的学习都是必不可少的 蛋白质。对这两组蛋白质的研究将增加我们的 能够构建适用于每一项的结构/功能关系 一组蛋白质。 对于我们的氧化还原研究，我们将重点关注两个结构良好的 表征每一类蛋白质的结构和机制 相似性：短链酰辅酶A脱氢酶(SCAD)和中链 黄素蛋白的酰辅酶A脱氢酶(MCAD)；核苷酸 还原酶(RNR)和甲烷单加氧酶羟基酶(MMO) 双核铁蛋白。电化学测量，突变的蛋白质， 和底物/产品类似物，以及特征明确的模型 系统，作为我们定义这些蛋白质结构的工具 支配电子的可行性和/或机制的特征 调职。 氧化还原数据提供了迄今为止最清晰的证据，证明电子 三种重要黄素蛋白的转移是热力学控制的 通过底物/产物结合。我们的氧化还原测量表明 底物/产物与MCAD和SCAD结合(Beta中的两个关键酶- 氧化)为电子提供热力学驱动力 转移反应。对双核铁蛋白的研究有 进展到了氧化还原数据在解决 电子传递机理和活性中心的特征 影响反应模式。氧化还原研究已经发挥了作用 对构成双核铁结构的关键作用 蛋白子宫铁蛋白及其抑制物作用机制的研究 结合到这种蛋白质上。有强有力的光谱证据表明 包括RNR和MMO在内的双核铁蛋白中的电子转移 受底物/组分结合的调节。

We are interested in determining how the protein environment affects the properties of a redox active center; we have chosen to study two classes of proteins containing two types of centers, flavoproteins and dinuclear iron proteins, both of which catalyze electron transfer reactions. Our goals are to relate the redox potentials to protein function, and determine how this function is further 'tuned' by substrate/component binding. Ultimately, we seek to identify the specific structural features of the active site which govern enzyme function and regulation. Members of both protein classes share common redox characteristics: 1) they have redox-active centers that participate in catalysis; 2) they are capable of one- or two-electron transfer, utilizing unique radical of mixed valent intermediates; 3) protonation accompanying electron transfer is important in thermodynamic regulation; and 4) their redox properties appear to be regulated by the binding of substrate, product, or regulatory protein components. The structures of members of both groups are either known by X-ray crystallography or are under intense study by other spectroscopic methods. The electrochemical data we are proposing to accumulate is essential to progress in the study of both classes of proteins. The investigation of both groups of proteins will increase our ability to construct structure/function relationships applicable to each set of proteins. For our redox studies, we will focus on two structurally well characterized proteins from each class with structural and mechanistic similarities: short chain acyl-CoA dehydrogenase (SCAD) and medium chain acyl-CoA dehydrogenase (MCAD) for the flavoproteins; ribonucleotide reductase (RNR) and methane monooxygenase hydroxylase (MMO) for the dinuclear iron proteins. Electrochemical measurements, mutated proteins, and substrate/product analogs, together with well characterized model systems, serve as our tools for defining those protein structural features which govern the feasibility and/or mechanism of electron transfer. Redox data has provided the clearest evidence to date that the electron transfer of three important flavoproteins is thermodynamically controlled by substrate/product binding. Our redox measurements have shown that substrate/product binding to both MCAD and SCAD (two key enzymes in beta- oxidation) provides the thermodynamic driving force for the electron transfer reaction. Studies on the dinuclear iron proteins have progressed to the point where redox data is critical in solving the mechanism of electron transport and the features of the active site which influence the mode of reactivity. Redox studies have already played a crucial role in contributing to the structure of the dinuclear iron protein uteroferrin and in modifying a proposed mechanism for inhibitor binding to this protein. There is strong spectroscopic evidence that electron transfer in dinuclear iron proteins, including RNR and MMO will be regulated by substrate/component binding.