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

在化学系生命过程化学项目的支持下，加州大学滨江分校的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位点转移到另一个生物位点，在代谢能量的产生和利用中发挥重要作用。 所谓的分叉黄素蛋白是一类除了黄素之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地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，能量上的上坡和下坡反应可以耦合在一起，大大增加了电子的还原能力。分叉的非凡能量学被很好地表征，但是关于这个过程的速率和机制，电子通过黄素蛋白分子的路径，我们知之甚少。 拟议的工作将导致对电子分叉过程的更深入的化学理解，电子分叉是生物体中一种重要的和进化上古老的能量守恒机制。 它也将导致识别的电子转移过程中，具体到分叉的定义属性。 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，并将纳入一个推广计划，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）分叉既有动力学方面，也有热力学方面，这是其生理功能的核心;（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。 实验方法将涉及一系列的快速反应动力学研究的巴豆酰辅酶A依赖的NADH：铁氧还蛋白氧化还原酶，ETF/bcd，从细菌Megaspaera elsdenii，专注于还原系统的NADH和其再氧化的高电位受体巴豆酰辅酶A和低电位受体，铁氧还蛋白。 反应后将进行停流光谱和冷冻淬灭EPR（电子顺磁共振）光谱的组合。所采取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，以探测分叉的机制。 我们的目标是探索电子转移过程中涉及黄素化学的内在分叉的元素。 希望这些研究将为生物学中最古老的进化节能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。

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.