BIOINORGANIC CHEMISTRY OF CARBON MONOXIDE DEHYDROGENASE

这项建议的长期目标是列举和 新型含镍、铁络合物和簇合物的表征 在来自热醋酸梭菌的一氧化碳脱氢酶中， 阐明它们在催化机制中所起的功能作用 这种酶。该酶催化两个主要反应，氧化 CO转化为CO2以及由CO，甲基， 和辅酶A。金属离子被组织成各种络合物，并 集群。所谓的镍铁络合物中的镍可以通过 双齿配体1，10-菲咯啉。移除会导致丢失 酶的合成酶活性，而不是其CO氧化活性。这个 镍可以通过添加Ni2+来重新插入和恢复活性 邻菲咯啉处理的酶。底物CO似乎结合在 不稳定的镍，表明它是合成酶的活性部位 活动。这是一项非同寻常的发现，将使 这种酶的作用机制将以意想不到的细节进行探索。 将对镍的移除和重新插入进行优化，以确定 可以拆卸和重新插入，以及它被绑得有多紧。这个 使邻菲罗啉配体在去除 镍将通过筛选各种配体进行评估 不同的结构特性。像Fe2+这样的外来金属可以 插入到不稳定的部位。它们的插入条件将是 进行优化。利用特定外来金属的性质， 配位配体的数目和性质以及配位配体的几何构型 不稳定的位置将使用各种光谱方法进行评估， 包括EPR、穆斯堡尔、EXAFS和核磁共振。酶的作用机制 将通过确定甲基底物是否与此结合来进行探测 不稳定的倪妮。 该酶的其他金属络合物的结构和功能 集群也将被评估。对一氧化碳使用抑制剂 氧化活性，将确定一氧化碳被氧化的位置 并以此为特征。它的氧化还原和光谱性质 将确定络合物和团簇。这些属性将是 纳入建立对该机制的更好理解 酶的活性。这项拟议的研究对 研究酶，因为镍是酶的一种不寻常的成分，以及 人们对它的生化作用知之甚少。

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.