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

该子项目是利用 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得主要资金， 因此可以在其他CRISP条目中表示。列出的机构是 中心，不一定是研究者的机构。 固氮酶的铁钼辅因子（FeMoco）上的二氮还原机制是生物无机化学中最大的谜团之一，最近的证据表明底物与铁结合。然而，固氮酶有许多铁原子，使其难以进行光谱分析。有一个迫切需要的一个和两个铁配合物具有相同的自旋状态和几何形状的固氮酶中的铁原子，作为光谱模型解释固氮酶的数据。我们的假设是FeMoco的还原活化产生用于底物结合和还原的瞬时不饱和铁位点。这些铁结合位点在配体贫乏的环境中具有高自旋铁，其几何形状与大多数已知的铁络合物中的几何形状完全不同，但与FeMoco铁位点相似。我们的初步证据表明，铁络合物的几何形状对其轨道能量有着主导性的影响，而轨道能量又决定了N2和其他小分子的结合强度。因此，拟议的研究重点是铁原子具有与FeMoco铁位点相似的几何结构（三角锥）、电子结构（具有弱场配体的高自旋）和光谱的化合物。这种不寻常的配位环境将通过将铁配位到天青蛋白脱辅基蛋白的突变体来加强，天青蛋白脱辅基蛋白已知将金属约束到三角金字塔几何形状。将通过ENDOR、红外、拉曼、穆斯堡尔和X射线吸收评价铁络合物。光谱结果将提供一个新的模型化合物的结构和固氮酶的已知数据之间的联系，和X射线吸收实验将显示的氧化水平，几何形状，和铁原子的结合模式。核磁共振的初步结果表明，在一些工程天青蛋白-铁复合物中，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.