Mechanism of carbon monoxide oxidation by the carbon monoxide dehydrogenase/acetyl-CoA synthase from Clostridium thermoaceticum: kinetic characterization of the intermediates.
Mechanism of carbon monoxide oxidation by the carbon monoxide dehydrogenase/acetyl-CoA synthase from Clostridium thermoaceticum: kinetic characterization of the intermediates.
复制标题
热乙酸梭菌的一氧化碳脱氢酶/乙酰辅酶A合酶氧化一氧化碳的机制：中间体的动力学表征。
DOI：
10.1021/bi970590m
复制
发表时间：
1997
期刊：
影响因子：
2.9
通讯作者：
Ragsdale,SW
中科院分区：
文献类型：
--
作者：
Seravalli,J;Kumar,M;Lu,WP;Ragsdale,SW
来自热乙酸梭菌的一氧化碳脱氢酶/乙酰辅酶A合酶（CODH/ACS）催化（i）由甲基化的类咕啉蛋白、CO和辅酶A合成乙酰辅酶A和（ii）将CO氧化成CO2。CO氧化发生在被称为簇C的含Ni和FeS的中心。电子从簇C转移到一个单独的金属中心，簇B，再转移到外部受体，如铁氧还蛋白。在这里描述的工作中，我们进行了还原滴定的CODH/ACS与CO和连二亚硫酸钠和监测的反应，通过电子顺磁共振（EPR）光谱。我们还进行了前稳态动力学研究快速冷冻淬火EPR光谱（EPR）和停流动力学。CODH/ACS的氧化还原滴定显示存在一个紫外可见和EPR沉默的电子受体，表示为中心S，它似乎与蛋白质中的任何其他金属中心无关。我们的结果支持了以前的建议[安德森，M。E、& Lindahl，P. A.（1994）Biochemistry 33，8702−8711;安德森，M. E、& Lindahl，P. A.（1996）Biochemistry 35，8371 - 8380]，团簇C的Cred 2形式比Cred 1形式多还原两个电子。滴定和预稳态研究的综合结果被用于制定CO氧化的机制，包括以下步骤：（i）CO结合到[Cred 1，Box，Xox]状态以产生Cred 1 −CO复合物;（ii）Cred 1到Cred 2的双电子还原与CO2释放一致;（iii）第二个CO分子结合到[Cred 2，Box，Xox]状态以形成Cred 2 −CO复合物;（iv）第二个CO分子与[Cred 2，Box，Xox]状态结合以形成Cred 2 −CO复合物。（iv）电子从Cred 2 −CO转移到团簇B，形成[Cred 2，Bred，Xred]，并协同释放第二个CO2。步骤iii与从Cred 2到Boxand Xox的内部电子转移竞争。在高CO浓度下，步骤iii是有利的，而在低浓度下，每个周转只有一个CO分子结合并经历氧化。催化循环的关闭涉及电子从还原酶转移到电子受体蛋白，如铁氧还蛋白。Xox是一种尚未表征的电子受体，可能是中心S还原的中间体。Cred 2状态似乎是集群C在稳态营业额的主要状态。第一个半反应的速率决定步骤是步骤iv，而在稳态周转期间，它似乎是电子转移到外部电子受体。
Carbon monoxide dehydrogenase/acetyl-CoA synthase (CODH/ACS) fromClostridium thermoaceticumcatalyzes (i) the synthesis of acetyl-CoA from a methylated corrinoid protein, CO, and coenzyme A and (ii) the oxidation of CO to CO2. CO oxidation occurs at a Ni- and FeS-containing center known as cluster C. Electrons are transferred from cluster C to a separate metal center, cluster B, to external acceptors like ferredoxin. In the work described here, we performed reductive titrations of CODH/ACS with CO and sodium dithionite and monitored the reaction by electron paramagnetic resonance (EPR) spectroscopy. We also performed pre-steady-state kinetic studies by rapid freeze−quench EPR spectroscopy (FQ-EPR) and stopped-flow kinetics. Redox titrations of CODH/ACS revealed the existence of a UV−visible and EPR-silent electron acceptor denoted center S that does not appear to be associated with any of the other metal centers in the protein. Our results support the previous proposals [Anderson, M. E., & Lindahl, P. A. (1994)Biochemistry33, 8702−8711; Anderson, M. E., & Lindahl, P. A. (1996)Biochemistry35, 8371−8380] that the Cred2form of cluster C is two electrons more reduced than the Cred1form. The combined results from titrations and pre-steady-state studies were used to formulate a mechanism for CO oxidation, composed of the following steps: (i) CO binding to the [Cred1,Box,Xox] state to yield a Cred1−CO complex; (ii) two-electron reduction of Cred1to Cred2concerted with CO2release; (iii) binding of a second CO molecule to the [Cred2,Box,Xox] state to form a Cred2−CO complex; (iv) electron transfer from Cred2−CO to cluster B to form [Cred2,Bred,Xred] with concerted release of the second CO2. Step iii competes with internal electron transfer from Cred2to Boxand Xox. At high CO concentrations, step iii is favored, whereas at low concentrations, only one CO molecule per turnover binds and undergoes oxidation. Closure of the catalytic cycle involves electron transfer from reduced enzyme to an electron acceptor protein, like ferredoxin. Xoxis a yet-uncharacterized electron acceptor that may be an intermediate in the reduction of center S. The Cred2state appears to be the predominant state of cluster C during steady-state turnover. The rate-determining step for the first half-reaction is step iv, while during steady-state turnover, it appears to be electron transfer to external electron acceptors.