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 蛋白质含有两种类型的中心，黄素蛋白和双核 铁蛋白，两者都催化电子转移反应。 我们 目标是将氧化还原电位与蛋白质功能联系起来， 确定该功能如何通过基板/组件进一步“调整” 约束力 最后，我们试图确定具体的结构 控制酶功能和调节的活性部位的特征。 这两种蛋白质类别的成员具有共同的氧化还原特征：1） 它们具有参与催化的氧化还原活性中心; 2）它们是 能够单或双电子转移，利用独特的自由基， 混合价中间体; 3）伴随电子转移的质子化 是重要的热力学调节;和4）他们的氧化还原性能 似乎受底物、产物或 调节蛋白组分。 两个群体成员的结构 要么是已知的X射线晶体学或正在紧张的研究， 其他光谱方法。 我们提出的电化学数据 积累是必不可少的进步，在研究这两个类的 proteins. 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个的结构/功能关系 一组蛋白质。 对于我们的氧化还原研究，我们将集中在两个结构良好的 从每个类别的结构和机制的特征蛋白质 相似性：短链酰基辅酶A脱氢酶（SCAD）和中链 黄素蛋白酰基辅酶A脱氢酶;核糖核苷酸 还原酶（RNR）和甲烷单加氧酶羟化酶（MMO）的活性。 双核铁蛋白 电化学测量，突变蛋白质， 和底物/产物类似物，以及良好表征的模型 系统，作为我们定义这些蛋白质结构的工具 控制电子的可行性和/或机制的特征 转移 氧化还原数据提供了迄今为止最清楚的证据， 三种重要的黄素蛋白的转移是受代谢控制的 通过底物/产物结合。 我们的氧化还原测量表明， 底物/产物结合MCAD和SCAD（β- 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 进展到氧化还原数据在解决 电子传递机制和活性中心的特点， 影响反应的方式。 氧化还原研究已经起到了 对双核铁的结构起着至关重要的作用 蛋白质子宫铁蛋白和修改抑制剂的建议机制 与这种蛋白质结合。 有强有力的光谱证据表明 双核铁蛋白中的电子转移，包括RNR和MMO， 通过底物/组分结合来调节。

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.