Nitrite Inhibition of Carbon Monoxide Dehydrogenase

醋酸盐发酵产生的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酯途径中的中心酶是一氧化碳脱氢酶(CODH)，它是一种复杂的金属酶，在厌氧环境中乙酸酯的合成和发酵中起着关键作用。在产甲烷的嗜热链霉菌中，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，然后被氧化成二氧化碳。本实验室的初步研究结果表明，亚硝酸盐(NO2-)对嗜热假单胞菌CODH的催化活性有抑制作用。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的机制。对抑制动力学和抑制酶的EPR性质的分析将使研究人员能够(I)提出CODH抑制亚硝酸盐的可能机制，以及(Ii)测试亚硝酸盐作为嗜热分枝杆菌CODH底物被还原的可能性。从这些研究中获得的信息将成为未来研究的基础，重点是阐明嗜热假单胞菌CodH的结构和机制，以及表征氮氧化物与金属酶的新的相互作用。

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.