BIOINORGANIC CHEMISTRY OF CARBON MONOXIDE DEHYDROGENASE

这项建议的长期目标是列举和 表征新的含镍和铁的络合物和簇合物， 在来自热乙酸梭菌的一氧化碳脱氢酶中， 阐明它们在催化机制中的功能作用， 酶 这种酶催化两种主要的反应， CO到CO2和从CO，一个甲基， 和辅酶A。 金属离子被组织成各种络合物， 集群 所谓的NiFe络合物中的镍可以通过 双齿配体1，10-菲咯啉。 移除导致 酶的合成酶活性，但不是在其CO氧化活性。 的 通过向反应体系中加入Ni ~（2+）， 菲咯啉处理的酶。 底物CO似乎结合在 不稳定的Ni，这表明它是合成酶的活性位点 活动 这是一个非凡的发现， 酶的作用机制将以意想不到的细节进行探索。 将优化Ni的去除和重新插入，以确定 可以被移除和重新插入，以及它被绑得有多紧。 的 这些属性使得菲咯啉配体在去除 通过筛选各种配体来评价镍， 不同的结构特性。 外来金属如Fe 2+可 插入到不稳定部位。 它们的插入条件将 优化。 利用特定的外来金属的特性， 配位配体的数量和性质，以及配位配体的几何形状。 将使用各种光谱方法评价不稳定位点， 包括EPR、Mossbauer、EXAFS和NMR。 酶的作用机制 将通过确定甲基底物是否与此结合来探测 不稳定镍。 酶的其他金属络合物的结构和功能， 还将对集群进行评估。 使用一氧化碳抑制剂 氧化活性，CO被氧化的位点将被确定 和表征了 的氧化还原性质和光谱性质。 将确定络合物和簇。 这些属性将 为更好地了解机制 的酶。 本文的研究对我国的农业生产具有普遍意义。 研究酶，因为镍是酶的一种不寻常的成分， 其生物化学作用知之甚少。

The long-term objectives for this proposal are to enumerate and characterize the novel nickel and iron-containing complexes and clusters in carbon monoxide dehydrogenase from Clostridium thermoaceticum, and to elucidate the functional roles they play in the catalytic mechanism of the enzyme. The enzyme catalyzes two major reactions, the oxidation of CO to CO2 and the synthesis of acetyl-coenzyme A from CO, a methyl group, and coenzyme A. The metal ions are organized into various complexes and clusters. the nickel in the so-called NiFe complex can be removed by the bidentate ligand 1,10-phenanthroline. Removal results in loss of the enzyme's synthase activity but not in its CO oxidation activity. The nickel can be reinserted and activity recovered by adding Ni2+ to the phenanthroline-treated enzyme. The substrate CO appears to bind at the labile Ni, suggesting that it is the active site for the synthase activity. This is an extraordinary discovery that will allow the mechanism of the enzyme to be probed in unexpected detail. Removal and reinsertion of Ni will be optimized, to determine how much can be removed and reinserted, and how tightly it is bound. The attributes which makes the phenanthroline ligand so effective in removing the nickel will be evaluated by screening a variety of ligands with different structural properties. Foreign metals such as Fe2+ can be inserted into the labile site. The conditions for their insertion will be optimized. Utilizing the properties of particular foreign metals, the number and nature of the coordinating ligands, and the geometry of the labile site will be evaluated using various spectroscopic methods, including EPR, Mossbauer, EXAFS, and NMR. The mechanism of the enzyme will be probed by determining whether the methyl substrate binds to this labile Ni. The structures and functions of the enzyme's other metal complex and clusters will be evaluated as well. Using an inhibitor to the CO oxidation activity, the site at which CO is oxidized will be determined and characterized. The redox and spectroscopic properties of the complexes and clusters will be determined. These properties will be incorporated into establishing a better understanding of the mechanism of the enzyme. The proposed research is of general significance to the study of enzymes, since nickel is an unusual constituent of enzymes, and its biochemical roles are poorly understood.