Nitrite Inhibition of Carbon Monoxide Dehydrogenase

醋酸发酵生成甲烷占环境中生物产生的甲烷的三分之二。产甲烷微生物利用乙酸的中心酶是一氧化碳脱氢酶（CODH），这是一种复杂的金属酶，在厌氧环境下醋酸的合成和发酵中起着关键作用。在产甲烷的嗜热古菌Methanosarcina中，CODH催化乙酰辅酶A的裂解，产生游离辅酶A、甲基和酶结合的羰基，羰基随后被氧化为二氧化碳。尽管CODH在生物甲烷生成和醋酸盐厌氧代谢中的重要性，但这种复杂金属酶的结构和功能之间的关系才刚刚开始被阐明。研究底物结合和CODH氧化机制的一个策略是检查酶与底物类似物和抑制剂的相互作用。本实验室的初步结果表明，亚硝酸盐（NO2-）抑制嗜热分枝杆菌CODH的催化活性。本研究项目将利用动力学分析和电子顺磁共振（EPR）光谱来研究亚硝酸盐抑制CODH的机理。该项目有三个具体目标：1。从抑制类型、可逆性程度、pH和酶氧化态的影响、底物和底物类似物的影响等方面分析亚硝酸盐对CODH的抑制动力学；利用EPR光谱鉴定CODH的五个已知金属中心中哪些受到亚硝酸盐的影响；说谎。确定亚硝酸盐是否可以作为CODH的新底物被还原，可能产生还原性氮化合物，如一氧化氮（NO）。这些实验的结果将把亚硝酸盐定义为一种新的CODH抑制剂，并将深入了解氮氧化物与酶的金属中心相互作用的本质。对抑制动力学和被抑制酶的EPR特性的分析将使研究人员能够(i)提出亚硝酸盐抑制CODH的可能机制，（ii）测试亚硝酸盐作为CODH的底物从嗜热分枝杆菌中还原的可能性。从这些研究中获得的信息将为今后的研究奠定基础，重点是阐明嗜热分枝杆菌CODH的结构和机制，以及表征氮氧化物与金属酶的新型相互作用。

Acetate fermentation to methane accounts for two-thirds of the methane produced biologically in the environment. The central enzyme in the pathway of acetate utilization by methane-producing microorganisms is carbon monoxide dehydrogenase (CODH), a complex metalloenzyme that plays a critical role in both the synthesis and fermentation of acetate in anaerobic environments. In the methanogenic archaeon Methanosarcina thermophila, CODH catalyzes the cleavage of acetyl coenzyme A to produce free coenzyme A, a methyl group, and an enzyme-bound carbonyl that is subsequently oxidized to carbon dioxide. Despite the importance of CODH in biological methane production and in the anaerobic metabolism of acetate, the relationships between the structure and function of this complex metalloenzyme are only beginning to be elucidated.One strategy for investigating the mechanism of substrate binding and oxidation by CODH is examining the interaction of the enzyme with substrate analogs and inhibitors. Preliminary results in this laboratory have demonstrated that nitrite (NO2-) inhibits the catalytic activity of CODH from M. thermophila. This research project will utilize kinetic analysis and electron paramagnetic resonance (EPR) spectroscopy to investigate the mechanism by which nitrite inhibits CODH. The project has three specific objectives:1. To analyze the kinetics of nitrite inhibition of CODH in terms of type of inhibition, extent of reversibility, effects of pH and enzyme oxidation state, and effects of substrates and substrate analogs;2. To utilize EPR spectroscopy to identify which of the five known metal centers of CODH are affected by nitrite; and3. To determine if nitrite can be reduced as a novel substrate for CODH, possibly producing a reduced nitrogen compound such as nitric oxide (NO).The results of these experiments will define nitrite as a new inhibitor of CODH and will provide insight into the nature of nitrogen oxide interactions with the metal centers of the enzyme. Analysis of the kinetics of inhibition and the EPR properties of the inhibited enzyme will enable the investigators to (i) propose a likely mechanism for nitrite inhibition of CODH and (ii) test the possibility that nitrite is reduced as a substrate for CODH from M. thermophila. Information derived from these studies will serve as a foundation for future research focusing on the elucidation of the structure and mechanism of CODH from M. thermophila and the characterization of novel interactions of nitrogen oxides with metalloenzymes.