Lu W P, Ragsdale S W
Department of Chemistry, University of Wisconsin, Milwaukee 53201.
J Biol Chem. 1991 Feb 25;266(6):3554-64.
The final steps in the synthesis of acetyl-CoA by CO dehydrogenase (CODH) have been studied by following the exchange reaction between CoA and the CoA moiety of acetyl-CoA. This reaction had been studied earlier (Pezacka, E., and Wood, H. G. (1986) J. Biol. Chem. 261, 1609-1615 and Ramer, W. E., Raybuck, S. A., Orme-Johnson, W. H., and Walsh, C. T. (1989) Biochemistry 28, 4675-4680). The CoA/acetyl-CoA exchange activity was determined at various controlled redox potentials and was found to be activated by a one-electron reduction with half-maximum activity occurring at -486 mV. There is approximately 2000-fold stimulation of the exchange by performing the reaction at -575 mV relative to the rate at -80 mV. Binding of CoA to CODH is not sensitive to the redox potential; therefore, the reductive activation affects some step other than association/dissociation of CoA. We propose that a metal center on CODH with a midpoint reduction potential of less than or equal to -486 mV is activated by a one-electron reduction to cleave the carbonyl-sulfur bond and/or bind the acetyl group of acetyl-CoA. Based on a comparison of the redox dependence of this reaction with that for methylation of CODH (Lu, W-P., Harder, S. R., and Ragsdale, S. W. (1990) J. Biol. Chem. 265, 3124-3133) and CO2 reduction and formation of the Ni-Fe-C EPR signal (Lindahl, P. A., Münck, E., and Ragsdale, S. W. (1990) J. Biol. Chem. 265, 3873-3879), we propose that the assembly of the acetyl group of acetyl-CoA, i.e. binding the methyl group of the methylated corrinoid/iron-sulfur protein, binding CO, and methyl migration to form the acetyl-CODH intermediate, occur at the novel Ni-Fe3-4-containing site in CODH. CO has two effects on the CoA/acetyl-CoA exchange: it activates the reaction due to its reductive capacity and its acts as a noncompetitive inhibitor. We also discovered that the CoA/acetyl-CoA exchange was inhibited by nitrous oxide via an oxidative mechanism. In the presence of a low-potential electron donor, CODH becomes a nitrous oxide reductase which catalytically converts N2O to N2. This study combined with earlier results (Lu, W-P., Harder, S. R., and Ragsdale, S. W. (1990) J. Biol. Chem. 265, 3124-3133) establishes that the two-subunit form of CODH is completely active in all reactions known to be catalyzed by CODH.
通过追踪辅酶A（CoA）与乙酰辅酶A（acetyl-CoA）的CoA部分之间的交换反应，对一氧化碳脱氢酶（CODH）合成乙酰辅酶A的最后步骤进行了研究。该反应 earlier（佩扎卡，E.，和伍德，H.G.（1986）《生物化学杂志》261，1609 - 1615以及拉默，W.E.，雷巴克，S.A.，奥姆 - 约翰逊，W.H.，和沃尔什，C.T.（1989）《生物化学》28，4675 - 4680）已有研究。在各种受控的氧化还原电位下测定了CoA/乙酰辅酶A交换活性，发现其通过单电子还原被激活，在 - 486 mV时出现半数最大活性。相对于 - 80 mV的速率，在 - 575 mV进行反应时，交换受到约2000倍的刺激。CoA与CODH的结合对氧化还原电位不敏感；因此，还原激活影响的是CoA缔合/解离之外的某个步骤。我们提出，CODH上一个中点还原电位小于或等于 - 486 mV的金属中心通过单电子还原被激活，以裂解羰基 - 硫键和/或结合乙酰辅酶A的乙酰基。基于该反应与CODH甲基化反应（卢，W - P.，哈德，S.R.，和拉格斯代尔，S.W.（1990）《生物化学杂志》265，3124 - 3133）以及CO2还原和Ni - Fe - C电子顺磁共振信号形成反应（林达尔，P.A.，明克，E.，和拉格斯代尔，S.W.（1990）《生物化学杂志》265，3873 - 3879）的氧化还原依赖性比较，我们提出乙酰辅酶A的乙酰基组装，即结合甲基化类咕啉/铁硫蛋白的甲基、结合CO以及甲基迁移形成乙酰 - CODH中间体，发生在CODH中含新型Ni - Fe3 - 4的位点。CO对CoA/乙酰辅酶A交换有两种作用：由于其还原能力激活反应，并且作为非竞争性抑制剂起作用。我们还发现一氧化二氮通过氧化机制抑制CoA/乙酰辅酶A交换。在低电位电子供体存在下，CODH成为一氧化二氮还原酶，催化将N2O转化为N2。本研究与早期结果（卢，W - P.，哈德，S.R.，和拉格斯代尔，S.W.（1990）《生物化学杂志》265，3124 - 3133）相结合，证实了CODH的二聚体形式在已知由CODH催化的所有反应中完全具有活性。
