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的二氮（N2）还原的分子机制。我们在这个提案中的目标是开发一种独特的实验方法，将能够直接检查底物减少。测量竞争性15 N动力学同位素效应（KIE）将允许检查氮键顺序沿着反应坐标的变化。这些实验可能使第一次直接调查的固氮酶复合物动力学级联内的二氮还原步骤。D20和D2效应（DKIEs）对15 N KIEs将使检查的还原质子化的N2和估计的内在KIEs。三重标记实验（15 N15 N/15 N14 N/14 N14 N）将进一步帮助阐明内在机制。实验结果将用于重新评估基于晶体结构的金属簇坐标的各种理论方法，并将支持或使提出的机理模型无效。长期计划包括研究各种形式（突变体）的酶与改变的反应位点环境和来自各种生物体和非酶催化剂的固氮酶。这项研究可能会导致一个新的和独特的理解固氮酶催化二氮还原的机制，并可能在固氮和金属酶领域的一般具有重要影响。

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.