And infrared spectroscopic evidence that a kinetically competent paramagnetic intermediate is formed when acetyl-coenzyme A synthase reacts with CO
And infrared spectroscopic evidence that a kinetically competent paramagnetic intermediate is formed when acetyl-coenzyme A synthase reacts with CO
复制标题
DOI：
10.1021/ja0528329
复制
发表时间：
2005-10-05
影响因子：
15
通讯作者：
Ragsdale, SW
中科院分区：
文献类型：
--
作者：
George, SJ;Seravalli, J;Ragsdale, SW
一氧化碳脱氢酶/乙酰辅酶a合成酶（CODH/ACS）是一种双功能酶，它使古细菌和细菌能够通过Wood - Ljundahl途径以CO和氢/二氧化碳自养生长。CODH还原二氧化碳产生的CO通过分子内通道转移到ACS的活性位点，与辅酶A和甲基阳离子结合生成乙酰辅酶A。ACS的活性位点包含一个由半胱氨酸硫原子桥接到双核中心的[4Fe−4S]簇。双核中心由两个镍原子组成，由两个不同的半胱氨酸硫桥接。附着在[4Fe−4S]上的Ni位点被称为近端Ni，而另一个Ni原子呈方形平面几何形状，被称为远端Ni。我们报道了高活性（0.67自旋/mol）异源表达单体ACS的羰基化形式的表征，通过快速冷冻淬火EPR （RFQ - EPR）和停止流动红外（SF - IR）光谱。ACS与CO的反应产生单一的金属-羰基物质，其形成速率（用SF - IR测量）与酶顺磁态（NiFeCspecies）的形成速率（用RFQ - EPR测量）相关。这些结果表明，当ACS与CO反应时，在溶液中观察到的主要形式是NiFeC种。NiFeC种含有+1氧化还原态的近端Ni和2+态的[4Fe−4S]簇，因此在活性羰基化形式中没有Ni(0)或Ni（II）态的证据。
Carbon monoxide dehydrogenase/acetyl-CoA synthase (CODH/ACS) is a bifunctional enzyme which enables archaea and bacteria to grow autotrophically on CO and hydrogen/carbon dioxide using the Wood−Ljundahl pathway. CO produced from reduction of carbon dioxide by CODH is transferred to the active site of ACS through an intramolecular tunnel, where it combines with Coenzyme A and a methyl cation to produce acetyl-CoA. The active site of ACS contains a single [4Fe−4S] cluster bridged by a cysteine sulfur atom to a binuclear center. The binuclear center is composed of two Ni atoms bridged by two separate cysteine sulfurs. The Ni site attached to the [4Fe−4S] is referred to as proximal Ni, while the other Ni atom, which assumes a square-planar geometry, is referred to as the distal site. We report the characterization of the carbonylated form of highly active (0.67 spins/mol) heterologously expressed monomeric ACS fromC.hydrogenoformansinE.coliby rapid-freeze quench EPR (RFQ−EPR) and stopped-flow infrared (SF−IR) spectroscopies. The reaction of ACS with CO produces a single metal−carbonyl species whose formation rate, measured by SF−IR, correlates with the rate of formation, measured by RFQ−EPR, of the paramagnetic state of the enzyme (NiFeCspecies). These results indicate that theNiFeCspecies is the predominant form observed in solution when ACS reacts with CO. The NiFeC species contains the proximal Ni in the +1 redox state and the [4Fe−4S] cluster in the 2+ state, thus there is no evidence for either a Ni(0) or a Ni(II) state in the active carbonylated form of the enzyme.