Biophysical Studies of Metalloenzymes

在化学系生命过程化学（CLP）项目的支持下，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金属生物化学的核心问题，金属生物化学是对生命基础的金属中心的研究。这些研究集中在进行生命反应的多金属金属酶及其合成类似物上。一个组成部分侧重于研究具有Fe4簇的酶和具有Fe3M， M = Fe或Mo的类似物。重点是这些簇的“有机金属”状态，它们具有Fe- c键。这种状态曾经被认为在生命中是罕见的，现在被认为是萜类生物合成酶的中间产物，并且被发现是世界上最大的金属酶超家族“自由基- sam （RS）”酶功能的中间产物，在所有形式的生命中发现了超过70万个成员，它们进行了惊人的多样性的基本反应。第二个组成部分将研究酶的同工酶的活性位点辅助因子，Fe7M, M = Mo， V，或Fe。就社会影响而言，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。最重要的是对学科和研究界的智力/科学贡献。支持这些的是对培养博士后、研究生和本科生的贡献，不仅在本小组，而且在合作者中。这个延伸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。电子顺磁共振(EPR)/电子核双共振（ENDOR）研究仿生合成[Fe3，M]；[4]3+ -烷基/烯烃/炔簇，M = Fe, Mo，有望加深对萜类生物合成中间体和RS酶的认识，与[Fe3，M；[S4], M = Fe和Mo簇提供了Mo在调节氮酶Fe7Mo催化辅因子性质中的作用的见解。此外，从程序中可以看出，这三种氮酶同工酶通过一个涉及十种状态的通用机制起作用，记为En， n = 0-8。n =偶态的mo -氮酶具有EPR活性，多数具有EPR和ENDOR特征。相比之下，V-和fe -氮酶的n =奇态是epr活性的，鉴于其机制的普遍性，它们的研究将使探索催化n =奇态成为可能。新的氮素酶同工酶结构将被用来探讨三种同工酶的反应性差异是来自不同异质金属的影响还是来自不同的同工酶活性位点环境。就广泛的科学影响而言，更好地了解氮酶是非常重要的。要记住的是，固氮酶进行生物固氮，将气态N2转化为两分子氨（氮的生物可用形式），世界上大约一半的人口依赖于固氮酶的固氮作用。该项目得到了数学和物理科学理事会化学司和生物科学理事会分子和细胞生物科学司分子生物物理集群的支持。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值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.