Bioinorganic Chemistry of Carbon Monoxide Dehydrogenase

描述(申请人提供)：这个项目的长期目标是了解乙酰辅酶A合成酶/一氧化碳脱氢酶(ACS/CODH)的催化机理，这是已知的最复杂的金属酶之一。这种对氧敏感的双功能酶含有两个镍-铁-硫簇活性中心，这在生物学上是独一无二的。它们由一条蛋白质隧道连接，通过该隧道，CO从一个位置迁移到另一个位置，受与催化机制错综复杂相关的构象变化控制。其中一个镍离子在还原活化过程中可能被还原为零价态，在催化过程中可能与甲基和乙酰基结合，因此该酶催化了生物上独有的有机金属反应机理。事实上，未来四年的两个具体目标是获得零价态和镍结合的甲基和乙酰加合物的直接物理证据。另一个目的是确定控制蛋白质构象的因素，并了解蛋白质构象是如何在催化作用下编排的。由于鲜为人知的原因，该酶是异质性的，因为在一个群体中只有~30%的ACS/CODH分子具有催化功能。第四个目标是确定这种异质性的来源，并在可能的情况下消除它。将使用一系列光谱方法，包括EPR和穆斯堡尔谱、核磁共振、X射线吸收和荧光。还将使用停流动力学和定点诱变。与公共卫生相关：艰难梭菌含有这种acs/codh酶，会导致抗生素相关性结肠炎、毒性巨结肠、肠穿孔，甚至导致人类死亡。我们对ACS/CODH的机制研究将有助于确定该酶在该病原体中所起的代谢作用，并确定防止艰难梭菌在肠道中增殖的策略。此外，ACS/CODH对环境健康也很重要，因为它可以从大气中去除CO，并从废弃的军事场所降解TNT。ACS/CODH参与了C1的代谢，它包含一个复杂的通道，CO通过该通道迁移，影响代谢通道的领域。许多其他金属酶在催化功能方面是不同的，这里描述的研究可能有助于阐明这种“半位点”反应的原因。

DESCRIPTION (provided by applicant): The long-term objective of this project is to understand the catalytic mechanism of acetyl-CoA synthase/carbon monoxide dehydrogenase (ACS/CODH), one of the most complex metalloenzymes known. This oxygen-sensitive bifunctional enzyme contains two nickel-iron-sulfur cluster active-sites which are unique in biology. They are connected by a protein tunnel through which CO migrates from one site to the other, controlled by a conformational change which is intricately correlated to the catalytic mechanism. One of the Ni ions may be reduced to a zero-valent state during reductive activation, and this ion may bind methyl and acetyl groups during catalysis; thus the enzyme catalyzes an organometallic reaction mechanism which is unique in biology. Indeed two specific aims for the next four years are to obtain direct physical evidence for the zero-valent state and for the Ni-bound methyl and acetyl adducts. Another aim is to determine the factors that control the conformation of the protein and to understand how protein conformation is choreographed with catalysis. For reasons that are poorly understood, the enzyme is heterogenous in that only ~ 30% of ACS/CODH molecules in a population are catalytically functional. A fourth aim is to determine the origin of this heterogeneity and eliminate it if possible. A cadre of spectrocopic methods will be used, including EPR and M"ssbauer spectroscopy, NMR, X-ray absorption, and fluoresence. Stopped-flow kinetics and site-directed mutagenesis will also be used. PUBLIC HEALTH RELEVANCE: Clostridium difficile, which contains this enzyme ACS/CODH, causes antibiotic-associated colitis, toxic megacolon, intestinal perforations and even death in humans. Our mechanistic study of ACS/CODH will help define the metabolic roles played by the enzyme in this pathogen, and identify strategies for preventing the proliferation of C. difficile in intestines. Also, ACS/CODH is important in environmental health, as it removes CO from the atmosphere and degrades TNT from abandoned military sites. ACS/CODH is involved in C1 metabolism and it contains a sophisticated tunnel through which CO migrates, impacting the field of metabolic channeling. A number of other metalloenzymes are heterogeneous in terms of catalytic function, and the studies described here might contribute to elucidating the reasons for such "half-sites" reactivity.