Biosynthesis of the Iron-Molybdenum Cofactor

该奖项是根据2009年美国复苏和再投资法(公法111-5)资助的。智力优点铁钼辅因子(FeMo-co，7Fe-9S-Mo-X-高柠檬酸)，发现于固氮酶的活性部位，是自然界最复杂的异金属辅因子之一，需要催化化学上困难的氮素(N_2)还原为氨(NH_3)。FeMo-co是在多种不同的蛋白质参与下，通过多步途径合成的，这些蛋白质连接了Fe、S和Mo。目前，途径中间体Fe-S团簇的确切结构尚不清楚，对于催化生物金属反应的酶如NiFB和NiFEN的分子机制更是知之甚少。通过结合遗传、生化和光谱技术，本研究旨在确定途径中间体的确切结构，并揭示执行多步骤生物金属反应的酶机制，这些反应是生物固氮的基础。主要的焦点将放在NifB及其产物NifB-co上，它们构成了FEMO-co生物合成途径的早期部分。NifB酶NifB-co以及同位素类似物将通过EPR、Mössbauer、EXAFS和NRVS光谱技术进行检测。这些光谱检查将得到生化分析的补充，以确定每个铁、S和间隙原子X的确切步骤(S)和结合方式。更广泛的影响所有生命都依赖于元素氮的输入，即通过FeMo-CO催化的生物固氮作用进入生物圈。了解FeMO-co生物合成途径中的酶如何执行生物金属化学可能会导致设计和合成新型催化剂，以改进Haber-Bosch化肥生产过程，从而降低农业能源成本。研究生和本科生都将在这项研究中发挥重要作用，并将接受广泛的跨学科技术培训。作为该项目的一部分，将开展一项针对初中生和高中生的外展活动。设计的活动将把生物化学带入中学课堂。

This award is funded under the American Recovery and Reinvestment Act of 2009 (Public Law 111-5).Intellectual Merit The iron-molybdenum cofactor (FeMo-co, 7Fe-9S-Mo-X-homocitrate), found within the active site of the enzyme nitrogenase, is among nature's most complex hetero-metallic cofactors and is required to catalyze the chemically difficult reduction of dinitrogen (N2) to ammonia (NH3). FeMo-co is synthesized by a multi-step pathway with the involvement of multiple different proteins that couple Fe, S and Mo. Currently, the exact structures of the pathway intermediate Fe-S clusters are unknown and even less is known regarding the molecular mechanisms of the enzymes such as NifB and NifEN that catalyze the bio-metallic reactions. By combined genetic, biochemical and spectroscopic techniques, this research aims to determine the exact structures of the pathway intermediates and uncover the enzymatic mechanisms that perform the multi-step bio-metallic reactions that are the foundation of biological nitrogen fixation. The major focus will be placed on NifB and its product, NifB-co, that form the early part of the FeMo-co biosynthetic pathway. The NifB enzyme, NifB-co, as well as isotopic analogs, will be examined by EPR, Mössbauer, EXAFS and NRVS spectroscopic techniques. These spectroscopic inspections will be complemented by biochemical analysis to determine the exact step(s) and mode of incorporation of each Fe, S and the interstitial atom X. Broader ImpactAll life depends on the input of the element nitrogen into the biosphere by biological nitrogen fixation catalyzed by FeMo-co. Understanding how the enzymes of the FeMo-co biosynthesis pathway perform the bio-metallic chemistry may lead to design and synthesis of novel catalysts to improve Haber-Bosch processes for fertilizer production resulting in a decrease in energy costs for agriculture. Both graduate and undergraduate students will have a major role in this research and will be trained in a wide range of cross-disciplinary techniques. As a part of this project, an outreach activity that targets junior and senior High School students will be performed. The designed activity will bring biochemistry into the secondary education classroom.