Spectroscopic and Kinetic Properties of the Molybdenum-containing, NAD+ - dependent Formate Dehydrogenase from Ralstonia eutropha
Spectroscopic and Kinetic Properties of the Molybdenum-containing, NAD+ - dependent Formate Dehydrogenase from Ralstonia eutropha
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DOI：
10.1074/jbc.m115.688457
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发表时间：
2016-01-15
影响因子：
4.8
通讯作者：
Hille, Russ
中科院分区：
文献类型：
--
作者：
Niks, Dimitri;Duvvuru, Jayant;Hille, Russ
我们研究了富营养罗氏菌含钼、依赖NAD(+)的FdsABG甲酸脱氢酶的快速反应动力学和光谱性质。我们证实了以前对该酶的稳态研究，并将其表征扩展到还原半反应(甲酸盐与氧化酶的反应)的快速动力学研究。我们还表征了钼中心在Mo-V状态下的电子顺磁共振信号，证明了在反应过程中底物Cα氢直接转移到钼中心。改变温度、微波功率和酶还原水平，我们能够清楚地识别酶的四个铁/硫簇的电子顺磁共振信号，并找到另外两个的提示性证据；我们观察到钼中心和其中一个铁/硫中心之间的磁相互作用，允许将这个信号分配给酶中特定的铁/硫簇。根据我们对钼中心结构的最新研究进展，我们提出了一个反应机理，它涉及从甲酸盐到钼中心的钼硫基团的直接氢化物转移。
We have examined the rapid reaction kinetics and spectroscopic properties of the molybdenum-containing, NAD(+) -dependent FdsABG formate dehydrogenase from Ralstonia eutropha. We confirm previous steady-state studies of the enzyme and extend its characterization to a rapid kinetic study of the reductive half-reaction (the reaction of formate with oxidized enzyme). We have also characterized the electron paramagnetic resonance signal of the molybdenum center in its Mo-V state and demonstrated the direct transfer of the substrate C alpha hydrogen to the molybdenum center in the course of the reaction. Varying temperature, microwave power, and level of enzyme reduction, we are able to clearly identify the electron paramagnetic resonance signals for four of the iron/sulfur clusters of the enzyme and find suggestive evidence for two others; we observe a magnetic interaction between the molybdenum center and one of the iron/sulfur centers, permitting assignment of this signal to a specific iron/sulfur cluster in the enzyme. In light of recent advances in our understanding of the structure of the molybdenum center, we propose a reaction mechanism involving direct hydride transfer from formate to a molybdenum-sulfur group of the molybdenum center.