Biophysical Studies of Metalloenzymes

在化学系生命过程化学(CLP)计划的支持下，西北大学的Brian Hoffman教授正在应用先进的顺磁共振技术来解决矿物生物化学中的核心问题，这是对生命基础的金属中心的研究。这些研究的重点是执行生命反应的多金属金属酶及其合成类似物。其中一个组分专注于研究具有Fe4簇的酶和具有Fe3M的类似物，M=Fe或Mo。重点放在这些团簇的有机金属状态上，它们以Fe-C键为特征。这种状态曾经被认为在生命中很罕见，现在被认为是萜类生物合成酶的中间体，并被发现是世界上最大的金属酶超家族--自由基-SAM(RS)酶功能的中心中间体，在所有生命形式中都发现了超过70万个成员，它们进行了惊人的多样化的基本反应。第二部分将研究固氮酶同工酶的活性部位辅因子，Fe7M，M=Mo，V，或Fe。在社会影响方面，对教学、培训和学习目标的反应可以被视为形成一个金字塔。处于顶端的是对该学科和研究界的智力/科学贡献。支持这些的是对博士后、研究生和本科生的培养做出的贡献，不仅在这个群体中，在合作者中也是如此。这一外联金字塔的一个关键组成部分是努力扩大对科学事业的参与，重点是妇女和代表性不足的少数民族。仿生合成[Fe3，M；S4]3+-烷基/烯烃/炔烃簇合物M=Fe，Mo的电子顺磁共振(EPR)/电子-核双共振(Endor)研究有望加强对萜类生物合成中间体和RS酶的了解，并与[Fe3，M；S4]，M=Fe和Mo簇合物进行比较，以深入了解Mo在调节固氮酶Fe7Mo催化辅因子的性质中的作用。此外，如本程序所示，这三种固氮酶同工酶通过一个涉及十个状态的通用机制发挥作用，表示为EN，n=0-8。钼固氮酶的n=偶数态是EPR活性的，大部分由EPR和Endor表征。相反，V-和Fe-固氮酶的n=奇态是EPR活性的，考虑到机理的普遍性，他们的研究将使探索催化n=奇态成为可能。固氮酶同工酶的新结构将被用来探索这三种同工酶之间的反应活性差异是来自不同的异金属的影响，还是来自不同的同工酶活性部位环境。就广泛的科学影响而言，更好地了解固氮酶是非常重要的。应该记住，固氮酶执行生物固氮，将气体氮气转化为两个分子的氨，氨是生物可用的氮的形式，世界上大约一半的人口依赖于固氮酶的固氮。这个项目得到了数学和物理科学局的化学部和生物科学局的分子和细胞生物科学部的分子生物物理学小组的支持。这个奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

With the support of the Chemistry of Life Processes (CLP) program in the Division of Chemistry, Professor Brian Hoffman of Northwestern University is applying advanced paramagnetic resonance techniques to the solution of central problems in metallobiochemistry, the study of metal centers fundamental to life. These studies focus on multimetallic metalloenzymes that carry out life’s reactions and their synthetic analogues. One component focuses on the study of enzymes with Fe4 clusters and analogues with Fe3M, M = Fe or Mo. The focus is on ‘organometallic’ states of these clusters, which feature an Fe-C bond. Once thought to be rare in life, such states are now proposed as intermediates in terpenoid-biosynthesis enzymes and are found as intermediates central to the function of the world’s largest superfamily of metalloenzymes, the ‘radical-SAM (RS)’ enzymes, with over 700,000 members identified throughout all forms of life, which carry out a spectacular diversity of essential reactions. A second component will investigate the active site cofactor of isozymes of the enzyme nitrogenase, Fe7M, M = Mo, V, or Fe. In terms of societal Impact, the response to aims of teaching, training, and learning can be viewed as forming a pyramid. At the apex are intellectual/scientific contributions to the discipline and to the research community. Supporting these are contributions to the training of postdocs, graduate students, and undergraduates, not only in this group but in those of collaborators. A critical component of this outreach pyramid is an effort to broaden participation in the scientific enterprise, with focus on women and underrepresented minorities.Electron paramagnetic resonance (EPR)/electron-nuclear double resonance (ENDOR) studies of biomimetic synthetic [Fe3,M;S4]3+–alkyl/alkene/alkyne clusters, M = Fe, Mo, are expected to enhance understanding of intermediates of terpenoid-biosynthesis and RS enzymes, with comparison of the [Fe3,M;S4], M = Fe and Mo clusters offering insights into the role of the Mo in modulating the properties of the nitrogenase Fe7Mo catalytic cofactor. Furthermore, as shown by this program, the three nitrogenase isozymes function through a universal mechanism involving ten states, denoted En, n = 0-8. The n = even states of Mo-nitrogenase are EPR active and a majority have been characterized by EPR and ENDOR. In contrast, the n = odd states of the V- and Fe-nitrogenases are EPR-active, and given the mechanistic universality, their study will enable probing the catalytic n = odd states. New constructs of the nitrogenase isozymes will be used to probe whether reactivity differences among the three isozymes derive from influences of the different heterometals or from the differing isozyme active-site environments. In terms of broad scientific impact, better understanding of the nitrogenase enzyme is of great importance. It is well to remember that the enzyme nitrogenase carries out biological nitrogen fixation, the conversion of gaseous N2 to two molecules of ammonia, the biologically usable form of nitrogen and that approximately half the world’s human population depends on nitrogen fixation by nitrogenase. This project is supported by the Division of Chemistry in the Directorate for Mathematical and Physical Sciences, and by the Molecular Biophysics cluster of the Division of Molecular and Cellular Biosciences in the Directorate for Biological Sciences.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.