Biological roles and mechanisms of nitric oxide reactions with iron-sulfur cluster transcriptional regulators

一氧化氮是一种有毒分子，由土壤细菌产生，在我们的身体中作为防御病原体试图建立感染。一氧化氮发挥其毒性作用的主要方式之一是通过与一组广泛存在的蛋白质反应，这些蛋白质含有一种由铁和硫组成的辅因子（称为铁硫簇）。这一组的成员在非常广泛的细胞过程中起着至关重要的作用。为了避免一氧化氮的毒性，致病（以及良性）细菌已经进化出了保护系统，其功能是通过化学反应将一氧化氮清除。铁-硫辅因子对一氧化氮特别敏感的事实已经在自然界中被利用，通过许多调节蛋白的进化，这些调节蛋白本身含有铁-硫簇，并作为生物开关发挥作用，在一氧化氮存在下开启细胞一氧化氮解毒反应。尽管铁硫簇合物与NO反应的重要性和广泛性，但对该反应过程知之甚少。该应用程序的重点是了解如何NO响应含铁硫簇调节器的功能。在这里，我们建议调查两个这样的监管机构（称为WhiD和NsrR）。其中一种（WhiD）是一个蛋白质家族的成员，仅在少数细菌中发现（包括结核分枝杆菌，结核病的病原体，世界上主要的杀手之一，以及天蓝色链霉菌，目前临床上使用的许多抗生素的来源）。已知该蛋白质家族的成员在这些细菌中与应激反应相关的细胞发育过程中发挥关键作用，并且对于M.结核病在人类宿主的不友好环境中存活多年，处于对抗生素具有高度耐药性的休眠状态。另一个（NsrR）是一个分布广泛但基本上未经研究的监管机构家族的成员。它作为一个主要的NO传感器通过控制细胞对NO毒性的反应。我们实验室最近的工作揭示了对这些调节蛋白质性质的重要新见解，包括第一次关于蛋白质结合的铁硫簇与一氧化氮反应的详细机械信息，导致形成以前未报道的产物。我们现在建议利用这些最新进展，探索，使用广泛的方法，NO与这些蛋白质的反应的生物化学。这将揭示前所未有的机制洞察NO传感调节蛋白的功能，并提供信息，将是所有铁硫蛋白NO反应的普遍重要性。

Nitric oxide is a poisonous molecule that is generated by soil bacteria and in our bodies as a defence against pathogenic organisms trying to establish infection. One of the major ways by which nitric oxide exerts its toxic effects is through reaction with a widespread group of proteins that contain a type of cofactor made from both iron and sulfur (called an iron-sulfur cluster). Members of this group play crucial roles in a very wide range of cellular processes. To avoid nitric oxide toxicity, disease-causing (as well as benign) bacteria have evolved protective systems that function to detoxify nitric oxide by removing it through chemical reaction. The fact that iron-sulfur cofactors are particularly sensitive to nitric oxide has been exploited in Nature, through the evolution of a number of regulatory proteins that themselves contain an iron-sulfur cluster and which function as biological switches, turning on the cellular nitric oxide detoxification response in the presence of nitric oxide. Despite the importance and widespread nature of the reaction of iron-sulfur clusters with NO, very little is known about this reaction process. This application is focussed on understanding how NO-responsive iron-sulfur cluster-containing regulators function. Here, we propose to investigate two such regulators (called WhiD and NsrR). One (WhiD) is a member of a family of proteins that are found only in a small number of bacteria (including Mycobacteria tuberculosis, the causative agent of tuberculosis, one of the world's major killers, and Streptomyces coelicolor, the source of many of the antibiotics currently in use in the clinic). Members of this protein family are known to play key roles in these bacteria in cell developmental processes associated with stress response, and are crucial for the ability of M. tuberculosis to survive in the inhospitable environment of a human host for years, in a dormant state that is highly resistant to antibiotics. The other (NsrR), is a member of a widely distributed but largely unstudied family of regulators. It functions as a primary NO sensor by controlling the cellular response to NO toxicity. Recent work in our laboratories has revealed important new insight into the nature of these regulatory proteins, including, for the first time, detailed mechanistic information about the reaction of a protein-bound iron-sulfur cluster with nitric oxide, leading to the formation of previously unreported products. We now propose to exploit these recent advances to explore, using a wide range of methods, the biochemistry of the reaction of NO with these proteins. This will reveal unprecedented mechanistic insight into how NO-sensing regulatory proteins function, and provide information that will be of general importance for all iron-sulfur protein NO reactions.