IRON-BOUND AZURIN AS MIMICS OF LOW COORDINATE IRON SITES OF NITROGENASES

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 氮素在固氮酶的铁钼辅因子(FeMoco)上的还原机理是生物无机化学的一大谜团，最近的证据表明底物与铁结合。然而，固氮酶含有大量的铁原子，这给光谱分析带来了困难。目前迫切需要一种与固氮酶中铁原子具有相同自旋状态和几何构型的一铁和两铁络合物作为光谱模型来解释固氮酶的数据。我们的假设是，FeMoco的还原活化产生了底物结合和还原的瞬时不饱和铁位。在缺乏配体的环境中，这些铁结合位具有高自旋铁，其几何构型与大多数已知的铁络合物完全不同，但与FeMoco铁位相似。我们的初步证据表明，铁络合物的几何形状对其轨道能量具有主导影响，而轨道能量反过来又决定了结合氮气和其他小分子的强度。因此，建议的研究集中在铁原子具有与FeMoco铁中心相似的几何结构(三角锥体)、电子结构(高自旋和弱场配体)和光谱的化合物。这种不寻常的配位环境将通过将铁与天青脱辅基蛋白的突变体配位来加强，这种突变体已知会将金属约束在三角金字塔几何图形上。铁的络合物将通过Endor、红外、拉曼、穆斯堡尔和X射线吸收进行评估。光谱结果将提供新模型化合物的结构与固氮酶的已知数据之间的联系，X射线吸收实验将显示铁原子的氧化程度、几何构型和结合模式。核磁共振的初步结果表明，在一些工程天青铁络合物中叠氮化物与铁原子结合，我们将使用X射线吸收来分析这种铁物种和其他新的铁物种。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The mechanism of dinitrogen reduction at the iron-molybdenum cofactor (FeMoco) of nitrogenase is one of the great mysteries of bioinorganic chemistry, and recent evidence suggests that substrates bind at iron. However, nitrogenase has many iron atoms, making it difficult to analyze spectroscopically. There is an urgent need for one- and two-iron complexes with the same spin state and geometry as the iron atoms in nitrogenase, to use as spectroscopic models for interpretation of the data on nitrogenase. Our hypothesis is that reductive activation of the FeMoco generates transient unsaturated iron sites for substrate binding and reduction. These iron binding sites would have highspin iron in a ligand-poor environment, with a geometry quite different from that in most known iron complexes but similar to the FeMoco iron sites. Our preliminary evidence shows that the geometry of an iron complex has a dominant influence on its orbital energies, which in turn determine the strength of binding N2 and other small molecules. Therefore, the proposed research focuses on compounds in which the iron atom has similar geometry (trigonal pyramidal), electronic structure (high-spin with weak-field ligands) and spectroscopy as the FeMoco iron sites. The unusual coordination environment will be enforced by coordinating iron to mutants of the azurin apo-protein, which is known to constrain metals to a trigonal pyramidal geometry. The iron complexes will be evaluated by ENDOR, infrared, Raman, M¿ssbauer, and x-ray absorption. The spectroscopic results will provide a link between the structures of novel model compounds and the known data for nitrogenase, and the x-ray absorption experiments will show the oxidation level,geometry, and binding modes to the iron atom. Preliminary results from NMR indicate binding of azide to the iron atom in some engineered azurin-iron complexes, and we will use x-ray absorption to analyze this and other novel iron species.