Mechanistic studies of nitrogenase catalysis

描述（由申请人提供）：该项目的目的是深入了解金属酶固氮酶催化二氮（N2）还原的分子机制。我们在此提案中的目标是开发一种独特的实验方法，能够直接检查底物的减少。测量竞争性 15N 动力学同位素效应 (KlEs) 将允许检查氮键顺序沿反应坐标的变化。这些实验可能能够首次直接研究固氮酶复合物动力学级联中的二氮还原步骤。 D20 和 D2 对 15N KIE 的影响 (D KIE) 将能够检查 N2 的还原质子化并估计内在 KIE。三重标记实验（15N15N /15N14N/14N14N）将进一步帮助阐明其内在机制。初步实验将使用来自Azotobacter vinelandii的酶进行，其晶体结构已确定。实验结果将用于重新评估基于该晶体结构的金属簇坐标的各种理论方法，并将支持或无效所提出的机械模型。长期计划包括研究反应位点环境改变的酶的各种形式（突变体）以及来自各种生物和非酶催化剂的固氮酶。这项研究可能会对固氮酶催化二氮还原的机制产生新的、独特的理解，并且可能对固氮和金属酶领域产生关键影响。

DESCRIPTION (provided by applicant): The objective of the project is to gain insight into the molecular mechanism of dinitrogen (N2) reduction catalyzed by the metalloenzyme nitrogenase. Our goal in this proposal is to develop a unique experimental methodology that will enable a direct examination of substrate reduction. Measuring competitive 15N kinetic isotope effects (KlEs) will allow examination of the change in nitrogen bond order along the reaction coordinate. These experiments may enable the first direct investigation of the dinitrogen reduction step within the nitrogenase complex kinetic cascade. D20 and D2 effects (D KIEs) on the 15N KIEs will enable examination of the reductive protonation of N2 and estimation of intrinsic KIEs. Triple labeling experiments (15N15N /15N14N/14N14N) will further assist the elucidation of the intrinsic mechanism.The initial experiments will be conducted with the enzyme from Azotobacter vinelandii whose crystal structure is available. The experimental findings will be used to reevaluate various theoretical approaches that were based on the metallic cluster coordinates from that crystal structure, and will support or invalidate proposed mechanistic models.The long-term plans include an investigation of various forms (mutants) of the enzyme with altered reaction site environments and nitrogenases from various organisms and non-enzymatic catalysts. This study may lead to a new and unique understanding of the mechanism by which nitrogenase catalyzes dinitrogen reduction and can have a critical impact in the field of nitrogen fixation and metalloenzymes in general.