Biophysical Studies of Metalloenzymes

金属酶的生物物理研究

• 批准号: 1908587
• 负责人: Brian Hoffman
• 金额: $ 90万
• 依托单位: Northwestern University
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-08-01 至 2024-07-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1908587&HistoricalAwards=false
• 关键词: Biophysical; Studies; Metalloenzymes

Enzymes, the catalysts for biological reactions, are able to efficiently perform very difficult chemical transformations. This project has two major long-range goals: (i) to help implement the conversion of cellulose, the major constituent of plant-derived biomass in nature, into green and sustainable biofuels; (ii) to understand how nature fertilizes the earth through the conversion of atmospheric nitrogen molecules into bioavailable nitrogen. Cellulose is potentially a renewable source of biofuels, but despite years of study, the efficient and economical degradation of cellulose remains a limiting factor in the industrial conversion of cellulose to biofuels. Polysaccharide monooxygenases are enzymes that use a copper active site to catalyze cellulose degradation, and their utilization could help to overcome the prohibitive costs of cellulose degradation. The reduction of nitrogen to yield two moles of ammonia, available as fertilizer, is catalyzed by the metalloenzyme, nitrogenase. The agronomic, economic, and social significance of nitrogenase can be appreciated by recognizing that it supplies approximately 50% of the N atoms in humans. The remainder is produced by the high-temperature/pressure Haber-Bosch industrial process, which uses H2 generated from CH4 as reductant and as a result produces vast amounts of CO2 and accounts for ~2% of the world's total energy consumption. The goal of this project is to understand the mechanism of these two important enzymes. This project will contribute to the training of graduate students and postdocs, with special attention to increasing the participation of women and underrepresented minorities in the scientific enterprise.This project is founded on techniques developed in the laboratory of the investigator for freeze-trapping reactive intermediates, reducing them in the frozen solid by radiolytic cryoreduction, and performing EPR/ENDOR characterizations of paramagnetic states. In the case of polysaccharide monooxygenases, a primary intermediate is freeze-trapped and paramagnetic one-electron reduced forms generated by cryoreduction. A step-annealing protocol propels this along its reaction pathway in a controlled fashion for study of successive intermediates that have only fleeting existence at ambient temperatures. This work is paralleled by studies of Cu-oxy, hydroperoxy, and peroxo model complexes, both with and without substrate present. The investigator and his group have established that nitrogenase functions by sequential accumulation of 4e-/4H+ to generate an activated state that undergoes reductive elimination of H2 coupled to N2 displacement and N≡N triple bond cleavage. The subsequent delivery of 4 additional electrons/protons then produces 2 NH3 as products to complete the catalytic cycle. In this project, the characterization of the nitrogenase mechanism by trapping intermediates through freeze-quench or cryoreduction with analysis by EPR/ENDOR, and by monitoring their interconversion, is sought.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.

酶是生物反应的催化剂，能够有效地进行非常困难的化学转化。 该项目有两个主要的长期目标：（i）帮助将自然界植物生物质的主要成分纤维素转化为绿色和可持续的生物燃料； (ii) 了解大自然如何通过将大气中的氮分子转化为生物可利用的氮来给地球施肥。 纤维素是潜在的生物燃料的可再生来源，但尽管经过多年的研究，纤维素的高效且经济的降解仍然是纤维素工业转化为生物燃料的限制因素。 多糖单加氧酶是利用铜活性位点催化纤维素降解的酶，它们的利用有助于克服纤维素降解的高昂成本。在金属酶、固氮酶的催化下，氮还原产生两摩尔氨，可用作肥料。通过认识到固氮酶提供了人类约 50% 的氮原子，可以认识到固氮酶的农艺、经济和社会意义。其余部分由高温/高压哈伯-博世工业流程生产，该流程使用 CH4 产生的 H2 作为还原剂，从而产生大量二氧化碳，约占世界总能源消耗的 2%。该项目的目标是了解这两种重要酶的机制。 该项目将有助于研究生和博士后的培训，特别关注增加妇女和代表性不足的少数群体在科学事业中的参与。该项目建立在研究人员实验室开发的冷冻捕获反应中间体技术的基础上，通过辐射解低温还原在冷冻固体中减少它们，并进行顺磁态的 EPR/ENDOR 表征。就多糖单加氧酶而言，主要中间体被冷冻捕获并通过冷冻还原产生顺磁性单电子还原形式。逐步退火方案以受控方式推动其沿反应途径前进，以研究在环境温度下仅短暂存在的连续中间体。这项工作与铜氧、氢过氧和过氧模型复合物的研究并行，无论有或没有底物存在。 研究人员和他的团队已经确定，固氮酶通过 4e-/4H+ 的连续积累产生激活状态，从而经历 H2 的还原消除以及 N2 置换和 N≡N 三键裂解。随后传递 4 个额外的电子/质子，然后产生 2 个 NH3 作为产物，完成催化循环。在该项目中，寻求通过冷冻淬灭或低温还原捕获中间体并通过 EPR/ENDOR 分析并监测其相互转化来表征固氮酶机制。该奖项反映了 NSF 的法定使命，并通过评估被认为值得支持利用基金会的智力优势和更广泛的影响审查标准。

项目成果

Hydrogen atom abstraction by a high-spin [FeIII=S] complex

高自旋 [FeIII=S] 配合物夺取氢原子

• DOI: 10.1016/j.chempr.2023.05.007
• 发表时间: 2023-05-01
• 期刊: Chem
• 影响因子: 23.5
• 作者: Juan A. Valdez;Duleeka C. Wannipurage;M. Pink;V. Carta;P. Moënne‐Loccoz;J. Telser;Jeremy M. Smith
• 通讯作者: Jeremy M. Smith

Characterization by ENDOR Spectroscopy of the Iron–Alkyl Bond in a Synthetic Counterpart of Organometallic Intermediates in Radical SAM Enzymes

通过 ENDOR 光谱表征自由基 SAM 酶中有机金属中间体的合成对应物中的铁烷基键

• DOI: 10.1021/jacs.2c07155
• 发表时间: 2022-09-28
• 期刊: Journal of the American Chemical Society
• 影响因子: 15
• 作者: Ho, Madeline B.;Jodts, Richard J.;Kim, Youngsuk;McSkimming, Alex;Suess, Daniel L. M.;Hoffman, Brian M.
• 通讯作者: Hoffman, Brian M.

A conformational equilibrium in the nitrogenase MoFe protein with an α-V70I amino acid substitution illuminates the mechanism of H 2 formation

固氮酶 MoFe 蛋白中α-V70I 氨基酸取代的构象平衡阐明了 H 2 形成的机制

• DOI: 10.1039/d2fd00153e
• 发表时间: 2023-07
• 期刊: Faraday Discussions
• 影响因子: 3.4
• 作者: Lukoyanov, Dmitriy A.;Yang, Zhi;Shisler, Krista;Peters, John W.;Raugei, Simone;Dean, Dennis R.;Seefeldt, Lance C.;Hoffman, Brian M.
• 通讯作者: Hoffman, Brian M.

Connecting the geometric and electronic structures of the nitrogenase iron–molybdenum cofactor through site-selective 57Fe labelling

通过位点选择性 57Fe 标记连接固氮酶铁钼辅因子的几何结构和电子结构

• DOI: 10.1038/s41557-023-01154-9
• 发表时间: 2023-05
• 期刊: Nature Chemistry
• 影响因子: 21.8
• 作者: Badding, Edward D.;Srisantitham, Suppachai;Lukoyanov, Dmitriy A.;Hoffman, Brian M.;Suess, Daniel L.
• 通讯作者: Suess, Daniel L.

The One-Electron Reduced Active-Site FeFe-Cofactor of Fe-Nitrogenase Contains a Hydride Bound to a Formally Oxidized Metal-Ion Core

Fe-固氮酶的单电子还原活性位点 FeFe-辅因子含有与形式氧化的金属离子核心结合的氢化物

• DOI: 10.1021/acs.inorgchem.2c00180
• 发表时间: 2022-04
• 期刊: Inorganic Chemistry
• 影响因子: 4.6
• 作者: Lukoyanov, Dmitriy A.;Harris, Derek F.;Yang, Zhi;Pérez;Dean, Dennis R.;Seefeldt, Lance C.;Hoffman, Brian M.
• 通讯作者: Hoffman, Brian M.