Lu W P, Jablonski P E, Rasche M, Ferry J G, Ragsdale S W
Department of Biochemistry, University of Nebraska, Lincoln 68583-0718.
J Biol Chem. 1994 Apr 1;269(13):9736-42.
Methanosarcina thermophila contains a multienzyme complex called the carbon-monoxide dehydrogenase complex, which has been resolved into a nickel/iron-sulfur and a corrinoid/iron-sulfur component. This complex plays a central role in acetoclastic methanogenesis. The Ni/Fe-S component catalyzes CO oxidation and has been proposed to be involved in cleavage of acetyl-CoA into its methyl, carbonyl, and CoA moieties. In the work reported here, three metal centers in the Ni/Fe-S component were characterized by electron paramagnetic resonance (EPR) spectroscopy and spectroelectrochemistry and pre-steady state kinetics. Center A contains nickel and iron and forms an EPR active adduct with CO, which is called the NiFeC species. The EPR spectrum of the NiFeC species has g values of 2.059, 2.051, and 2.029 and is observable at temperatures as high as 150 K. This signal had previously been observed only in the carbon-monoxide dehydrogenase complex of M. thermophila and the acetyl-CoA synthase from acetate-producing bacteria. Incubation of the CO-reduced Ni/Fe-S component with acetyl-CoA resulted in an increase in intensity of the NiFeC signal, which supports a role for the component in the cleavage of acetyl-CoA. Generation of the NiFeC EPR signal occurs with a rate constant of 0.4 s-1, a result that demonstrates the kinetic competence of this species in the acetyl-CoA cleavage reaction but rules it out as the site of oxidation of CO to CO2. Center B is likely to be a [4Fe-4S]2+/1+ center with g values of 2.04, 1.93, and 1.89 (gav = 1.95) and a standard reduction potential (E'0) of -444 mV. At potentials less than -500 mV, another EPR signal develops that appears to originate from another state of Center B. Center C is a fast relaxing center with g values of 2.02, 1.88, and 1.71 (gav = 1.87) and an E'0 of -154 mV.
嗜热甲烷八叠球菌含有一种称为一氧化碳脱氢酶复合物的多酶复合体，该复合体已被解析为镍/铁硫和类咕啉/铁硫组分。这种复合体在乙酸裂解产甲烷过程中起着核心作用。镍/铁硫组分催化一氧化碳氧化，并且有人提出它参与将乙酰辅酶A裂解为其甲基、羰基和辅酶A部分。在本文报道的工作中，通过电子顺磁共振（EPR）光谱、光谱电化学和预稳态动力学对镍/铁硫组分中的三个金属中心进行了表征。中心A含有镍和铁，并与一氧化碳形成一种EPR活性加合物，称为NiFeC物种。NiFeC物种的EPR光谱的g值为2.059、2.051和2.029，在高达150 K的温度下都可观测到。这种信号以前仅在嗜热甲烷八叠球菌的一氧化碳脱氢酶复合体和产乙酸细菌的乙酰辅酶A合成酶中观察到。用乙酰辅酶A孵育一氧化碳还原的镍/铁硫组分导致NiFeC信号强度增加这一结果支持了该组分在乙酰辅酶A裂解中的作用。NiFeC EPR信号的产生速率常数为0.4 s-1，这一结果证明了该物种在乙酰辅酶A裂解反应中的动力学活性，但排除了它作为一氧化碳氧化为二氧化碳的位点的可能性。中心B可能是一个[4Fe-4S]2+/1+中心，g值为2.04、1.93和1.89（gav = 1.95），标准还原电位（E'0）为 -444 mV。在电位小于 -500 mV时，会出现另一种EPR信号，似乎起源于中心B的另一种状态。中心C是一个快速弛豫中心，g值为2.02、1.88和1.71（gav = 1.87），E'0为 -154 mV。
