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.

在化学系生命过程化学项目的支持下，来自加州大学河滨分校的Russ Hille教授将研究一种名为EtfAB/BCD的黄素蛋白的行为。黄素，如维生素核黄素，通过介导电子从一个生物部位到另一个生物部位的转移，在代谢能量的产生和利用中发挥着重要作用。所谓的分叉黄素蛋白是一类除黄素外还含有多个这样的位点的黄素蛋白。在分叉中，电子对一次一个电子传递到两个不同的位置。这提供了一种可行的方法，通过这种方法，能量上行和下行反应可以结合在一起，大大增加电子的还原能力。人们很好地描述了分叉的非凡能量学，但关于这个过程的速度和机制，即电子通过黄素蛋白分子的路径，人们知之甚少。这项拟议的工作将导致对电子分叉过程的更深层次的化学理解，电子分叉是生物体内能量守恒的一种基本的重要和进化上古老的机制。它还将导致识别特定于分叉的电子转移过程的定义属性。这项工作将为研究生提供各种光谱和反应速率测量方面的广泛培训，并被整合到一个扩展计划中，为高中教师、学生和大学本科生提供这些领域的研究经验。拟议工作的中心假设是：(1)分叉具有动力学和热力学方面，这是其生理功能的中心；(2)质子化/去质子化事件在调节与分叉相关的电子转移事件中发挥关键作用。实验方法将涉及一系列快速反应动力学研究依赖于巴豆基辅酶A的NADH：铁氧还蛋白氧化还原酶，ETF/BCD，从细菌Megasphaera elsdenii，重点是NADH还原系统和它的再氧化由高电位受体巴豆酰-CoA和低电位受体铁氧还蛋白。在反应之后，将结合停流光谱和冷冻-淬灭EPR(电子顺磁共振)光谱。所采取的实验方法包括广泛的快速动力学技术，包括探索分叉机制的溶剂同位素研究。我们的目标是探索涉及黄素化学的电子转移过程中的元素，这些元素是分叉的内在因素。希望这些研究将为生物中最古老的能源节约机制之一提供基本的见解。该奖项反映了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。