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：㈠帮助将纤维素-自然界植物生物量的主要组成部分-转化为绿色和可持续的生物燃料; ㈡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使地球肥沃。 纤维素是生物燃料的潜在可再生来源，但尽管经过多年的研究，纤维素的有效和经济降解仍然是纤维素向生物燃料的工业转化的限制因素。 多糖单加氧酶是利用铜活性位点催化纤维素降解的酶，并且它们的利用可以帮助克服纤维素降解的高昂成本。氮的还原产生两摩尔氨，可用作肥料，是由金属酶，固氮酶催化的。固氮酶的农学、经济和社会意义可以通过认识到它提供了人类大约50%的N原子来理解。其余的是由高温/高压哈伯-博世工业过程生产的，该过程使用由CH 4产生的H2作为还原剂，因此产生大量的CO2，占世界总能耗的约2%。该项目的目标是了解这两种重要酶的机制。 该项目将促进研究生和博士后的培训，特别注意增加妇女和代表人数不足的少数民族在科学事业中的参与，该项目的基础是研究人员实验室开发的冷冻捕获活性中间体的技术，通过辐射分解冷冻还原法在冷冻固体中还原它们，并对顺磁状态进行EPR/ENDOR表征。在多糖单加氧酶的情况下，初级中间体是冷冻捕获的顺磁性单电子还原形式，其通过冷冻还原产生。一个步骤退火协议推动这沿着其反应途径，在一个受控的方式研究连续的中间体，只有短暂的存在，在环境温度下。这项工作是通过研究铜氧，氢过氧，过氧模型配合物，无论有和没有基板存在。 研究者和他的小组已经确定，固氮酶的功能是通过4 e-/4 H+的连续积累来产生活化状态，该活化状态经历H2的还原消除，再加上N2置换和N#8801;N三键断裂。随后传递4个额外的电子/质子，然后产生2个NH3作为产物以完成催化循环。在这个项目中，固氮酶的机制，通过捕获中间体，通过冷冻淬火或冷冻还原与EPR/ENDOR分析，并通过监测它们的相互转化的表征，是searched.This奖项反映了NSF的法定使命，并已被认为是值得通过使用基金会的智力价值和更广泛的影响审查标准进行评估的支持。

项目成果

Heteroleptic Square Planar Cobalt(I/II) Complexes

杂配方平面钴(I/II)配合物

• DOI: 10.1002/ejic.202200675
• 发表时间: 2023
• 期刊: European Journal of Inorganic Chemistry
• 影响因子: 2.3
• 作者: Gao, Yafei;Lee, Wei‐Tsung;Carta, Veronica;Chen, Chun‐Hsing;Telser, Joshua;Smith, Jeremy M.
• 通讯作者: Smith, Jeremy 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
• 期刊: Faraday Discussions
• 影响因子: 3.4
• 作者: Lukoyanov, Dmitriy A.;Yang, Zhi-Yong;Shisler, Krista;Peters, John W.;Raugei, Simone;Dean, Dennis R.;Seefeldt, Lance C.;Hoffman, Brian M.
• 通讯作者: Hoffman, Brian M.

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
• 期刊: Inorganic Chemistry
• 影响因子: 4.6
• 作者: Lukoyanov, Dmitriy A.;Harris, Derek F.;Yang, Zhi-Yong;Pérez-González, Ana;Dean, Dennis R.;Seefeldt, Lance C.;Hoffman, Brian M.
• 通讯作者: Hoffman, Brian M.

Computational Description of Alkylated Iron–Sulfur Organometallic Clusters

烷基化铁硫有机金属簇的计算描述

• DOI: 10.1021/jacs.3c03062
• 发表时间: 2023
• 期刊: Journal of the American Chemical Society
• 影响因子: 15
• 作者: Jodts, Richard J.;Wittkop, M;Ho, Madeline B.;Broderick, William E.;Broderick, Joan B.;Hoffman, Brian M.;Mosquera, Martín A.
• 通讯作者: Mosquera, Martín A.

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

• DOI: 10.1016/j.chempr.2023.05.007
• 发表时间: 2023-09-14
• 期刊: CHEM
• 影响因子: 23.5
• 作者: Valdez-Moreira，Juan A.;Wannipurage，Duleeka C.;Smith，Jeremy M.
• 通讯作者: Smith，Jeremy M.