Biophysical Studies of Metalloenzymes

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：(1)帮助实现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；(2)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氮来使地球肥沃。纤维素是一种潜在的可再生生物燃料来源，但尽管经过多年的研究，纤维素的高效和经济降解仍然是限制纤维素工业转化为生物燃料的一个因素。多糖单加氧酶是一种利用铜的活性中心来催化纤维素降解的酶，利用它们可以帮助克服纤维素降解的高昂成本。氮气还原成两摩尔氨，可用作肥料，由金属酶--固氮酶催化。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原子来实现。其余的由高温/高压Haber-Bosch工业过程产生，该过程使用CH4产生的H2作为还原剂，结果产生大量二氧化碳，占世界总能源消耗的约2%。该项目的目标是了解这两种重要酶的作用机制。该项目将有助于培养研究生和博士后，特别注意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技术的基础上，这些技术用于冷冻捕获活性中间体，通过辐射分解低温还原将其减少在冷冻固体中，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，并通过低温还原产生顺磁性的单电子还原形式。为了研究在环境温度下只有短暂存在的连续中间体，步进式退火法以受控的方式推动这一过程。这项工作与铜氧基、过氧化氢和过氧基模型络合物的研究是平行的，有底物存在和没有底物存在。这位研究人员和他的团队已经确定，固氮酶的功能是通过顺序积累4E-/4H+来产生一种活化状态，该活化状态经历了H2的还原消除、氮的置换和N&Amp；N的三键断裂。随后提供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，通过冷冻-淬火或冷冻还原通过EPR/Endor分析捕获中间体，并通过监测它们的相互转化来表征固氮酶机制。这一奖项反映了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。

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.