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：(i)帮助将纤维素（自然界中植物来源的生物质的主要成分）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的氨，可用作肥料，是由金属酶，即氮酶催化的。人类体内约50%的N原子由氮酶提供，因此我们可以认识到氮酶在农艺、经济和社会方面的重要意义。其余的由高温/高压Haber-Bosch工业流程生产，该流程使用由CH4产生的H2作为还原剂，因此产生大量的二氧化碳，占世界总能耗的2%。该项目的目标是了解这两种重要酶的作用机制。这个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民族对科学事业的参与。该项目建立在研究员实验室开发的冷冻捕获活性中间体技术的基础上，通过辐射裂解冷冻还原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，初级中间体被冷冻捕获，并通过冷冻还原产生顺磁性单电子还原形式。阶梯式退火方案以受控的方式沿着其反应途径推进，用于研究在环境温度下仅短暂存在的连续中间体。这项工作与有底物和无底物存在的cu -氧、氢过氧和过氧模型配合物的研究是平行的。研究者和他的团队已经确定，氮酶的功能是通过4e-/4H+的顺序积累来产生激活状态，经过H2的还原消除，再加上N2的位移和N&amp；#8801；N三键解理。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。在这个项目中，通过EPR/ENDOR分析，通过冷冻或冷冻还原捕获中间体，并通过监测它们的相互转化，寻求对氮酶机制的表征。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。

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.