The Crotonyl-CoA-dependent NADH:ferredoxin Oxidoreductase from M. Elsdenii

巴豆酰辅酶 A 依赖性 NADH：来自 M. Elsdenii 的铁氧还蛋白氧化还原酶

• 批准号: 2101672
• 负责人: Russ Hille
• 金额: $ 45万
• 依托单位: University of California-Riverside
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-08-01 至 2024-07-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=2101672&HistoricalAwards=false
• 关键词: Crotonyl; CoA; dependent; NADH; ferredoxin

With the support of the Chemistry of Life Processes Program in the Division of Chemistry, Professor Russ Hille from the University of California, Riverside will examine the behavior of a flavoprotein known as EtfAB/bcd. Flavins, such as the vitamin riboflavin, play important roles in the generation and utilization of metabolic energy by mediating the transfer of electrons from one biological site to another. So-called bifurcating flavoproteins are a class of flavoproteins containing multiple such sites in addition to their flavins. In bifurcation, electron pairs are delivered one electron at a time to two different sites. This provides a viable means by which energetically uphill and downhill reactions can be coupled together greatly increasing the reducing power of the electrons. The extraordinary energetics of bifurcation are well characterized, but little is known regarding the rates of this process and the mechanism, the paths that the electrons follow through the flavoprotein molecules. The proposed work will lead to a deeper chemical understanding of the process of electron bifurcation, a fundamentally important and evolutionarily ancient mechanism of energy conservation in living organisms. It will also lead to the identification of the defining properties of the electron-transfer processes that are specific to bifurcation. The work will provide graduate students with extensive training in a variety of spectroscopic and reaction rate measurements and is integrated into an outreach program to provide high school teachers, students and college undergraduates with research experience in these areas.The central hypotheses of the proposed work are that: (1) there is a kinetic as well as a thermodynamic aspect to bifurcation that is central to its physiological function; and (2) that protonation/deprotonation events play a critical role in modulation the electron transfer events associated with bifurcation. The experimental approach will involve a range of rapid reaction kinetic studies of the crotonylCoA-dependent NADH:ferredoxin oxidoreductase, ETF/bcd, from the bacterium Megasphaera elsdenii, focusing on the reduction of the system by NADH and its reoxidation by the high-potential acceptor crotonyl-CoA and the low-potential acceptor, ferredoxin. The reactions will be followed by a combination of stopped-flow spectroscopy and freeze-quench EPR (electron paramagnetic resonance) spectroscopy. The experimental approach taken includes a wide range of rapid kinetic techniques, including solvent isotope studies to probe the mechanism of bifurcation. The goal is to probe the elements of electron-transfer processes involving flavin chemistry that are intrinsic to bifurcation. It is hoped that these studies will provide fundamental insights into one of the most evolutionarily ancient mechanisms for energy conservation in biology.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.

在化学过程中的化学过程中的支持下，加利福尼亚大学的Russ Hille教授将研究一种称为ETFAB/BCD的黄蛋蛋白的行为。 黄素（例如维生素核黄素）通过介导电子从一种生物位点传递到另一种生物学位，在代谢能的产生和利用中起重要作用。 所谓的分叉黄蛋蛋白是一类黄酮蛋白，除了黄素外，还含有多种此类部位。 在分叉中，将电子对一次传递到两个不同的位点。 这提供了一种可行的手段，可以将能量上坡和下坡反应耦合在一起，从而大大增加电子的还原能力。分叉的非凡能量学的特征是，关于该过程的速率和机制，电子通过黄素分子遵循的路径知之甚少。 拟议的工作将导致对电子分叉过程的更深入的化学理解，这是一种从根本上重要而进化的古老的生物节能机制。 它还将导致鉴定特定于分叉的电子转移过程的定义特性。 这项工作将为研究生提供各种光谱和反应率测量的广泛培训，并将其纳入外展计划，以在这些领域为高中教师，学生和大学的大学生提供研究经验。拟议工作的中心假设是那是：（1）分叉的动力学和热力学方面，对其生理功能至关重要； （2）质子化/去质子化事件在调制与分叉相关的电子转移事件中起关键作用。 实验方法将涉及依赖Crotonylcoa依赖性NADH的一系列快速反应动力学研究：铁雷蛋白氧化还原酶ETF/BCD，来自Megasphaera Elsdenii细菌，重点是NADH的减少及其对系统的降低，并由高利用率接受者重新氧化。 Crotonyl-COA和低电势受体Ferredoxin。 反应将结合停止流光谱和冻结EPR（电子顺磁共振）光谱的组合。采用的实验方法包括广泛的快速动力学技术，包括溶剂同位素研究，以探测分叉的机理。 目的是探测涉及黄素化学的电子转移过程的元素，这些过程与分叉固有。 希望这些研究能够为生物学中最古老的能源保护机制之一提供基本的见解。该奖项反映了NSF的法定任务，并被认为是通过基金会的知识分子和更广泛影响的评估标准来评估的值得支持的。