Mechanistic studies of nitrogenase catalysis

描述(申请人提供)：该项目的目标是深入了解金属酶固氮酶催化氮气还原的分子机制。我们在这项提案中的目标是开发一种独特的实验方法，使直接检查底物还原成为可能。测量竞争性15N动力学同位素效应(KLES)将允许检查沿反应坐标的氮键顺序的变化。这些实验可能使首次直接研究固氮酶复合体动力学级联中的氮还原步骤。对15N Kie的D20和D2效应(DKie)将使研究N2的还原质子化和估计本征Kie成为可能。三重标记实验(15N15N/15N14N/14N14N)将进一步帮助阐明其内在机制。初步实验将以来自棕色固氮菌的酶进行，该酶的晶体结构是可用的。这些实验结果将被用来重新评估基于该晶体结构的金属团簇坐标的各种理论方法，并将支持或无效所提出的机理模型。长期计划包括研究不同形式(突变体)的酶，改变反应场所环境，以及来自不同生物体和非酶催化剂的固氮酶。这项研究可能会导致对固氮酶催化氮素还原的机制有一个新的和独特的理解，并可能在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.