Collaborative Research: Chemical and Genetic Modification of Biological Iron-Sulfur Clusters: Azotobacter Vinelandii Fereedoxin I

铁硫(Fe-S)蛋白含有主要由Fe、S2-和半胱氨酸残基组成的簇。这些蛋白质普遍存在于所有已知的生物体中，是每一种基本生物过程的中心。分别含有1，2和4个Fe原子的三种类型的团簇已经得到了很好的表征，最近发现了含有三个Fe原子的团簇。前三种表现出多个氧化水平。最近的研究表明，电子传递并不是这些蛋白质的唯一功能。现在非常清楚的是，Fe-S团簇结构的多样性比以前认识的要多得多，并且还在继续扩大。这种化学远比简单、可逆单电子氧化还原行为广泛。这项建议的目标是确定初级序列如何确定在给定蛋白质中哪种类型的簇具有功能。为了实现这一目标，我们将深入研究棕色拟青霉中蛋白结合的3-Fe和4-Fe(Fe-S)簇的结构和反应活性。这种小蛋白质非常适合这样的研究，因为它表达多种形式，X射线数据正在变得可用。因此，计划提纯和结晶这三种新形式的AvFdI，对其进行物理化学表征，通过定点突变对其进行修饰，并对所产生的菲多辛进行纯化和表征。这项工作可能对我们理解固氮酶和其他含有铁-S中心的多中心氧化还原蛋白至关重要。强烈建议优先提供支持。

Iron sulfur (Fe-S) proteins contain clusters composed predominately of Fe, S2- and cysteine residues. These proteins are ubiquitous occuring in all known organisms and are central to every essential biological process. Three types of clusters containing respectively, 1, 2, and 4 Fe atoms are now well characterized and recently clusters containg three Fe atoms have been discovered. The first three exhibit multiple oxidation levels. It has recently been shown that electron transport is not the sole function of these proteins. It is now abundantly clear that the variety of Fe-S cluster structures is greater than previously recognized and continues to expand. The chemistry is far more extensive than simple, reversible one-electron redox behavior. The goal of this proposal is to ascertain how the primary sequence determines which type of cluster is functional in a given protein. To achieve this goal the structures and reactivities of protein-bound 3 FE and 4 FE (Fe-S) clusters in A. vinelandii fereedoxin I (AVFD) will be studied in depth. This small protein is ideally suited for such an investigation, as it expresses multiple forms and X- ray data is becoming available. Consequently, it is planned to purify and crystallize the three new forms of AvFdI, characterize them physico-chemically, modify them by site directed mutagenesis, and purify and characterize the resulting fereedoxins. This work may prove crucial to our quest to understand nitrogenase and other multicentered redox proteins containing Fe-S centers. Support is strongly recommended with a high priority.