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Crystallographic and kinetic analyses of the FdsBG subcomplex of the cytosolic formate dehydrogenase FdsABG from Cupriavidus necator.
Journal of Biological Chemistry ( IF 5.5 ) Pub Date : 2020-04-05 , DOI: 10.1074/jbc.ra120.013264
Tynan Young 1 , Dimitri Niks 1 , Sheron Hakopian 1 , Timothy K Tam 1 , Xuejun Yu 1 , Russ Hille 2 , Gregor M Blaha 2
Affiliation  

Formate oxidation to carbon dioxide is a key reaction in one-carbon compound metabolism, and its reverse reaction represents the first step in carbon assimilation in the acetogenic and methanogenic branches of many anaerobic organisms. The molybdenum-containing dehydrogenase FdsABG is a soluble NAD+-dependent formate dehydrogenase and a member of the NADH dehydrogenase superfamily. Here, we present the first structure of the FdsBG subcomplex of the cytosolic FdsABG formate dehydrogenase from the hydrogen-oxidizing bacterium Cupriavidus necator H16 both with and without bound NADH. The structures revealed that the two iron-sulfur clusters, Fe4S4 in FdsB and Fe2S2 in FdsG, are closer to the FMN than they are in other NADH dehydrogenases. Rapid kinetic studies and EPR measurements of rapid freeze-quenched samples of the NADH reduction of FdsBG identified a neutral flavin semiquinone, FMNH•, not previously observed to participate in NADH-mediated reduction of the FdsABG holoenzyme. We found that this semiquinone forms through the transfer of one electron from the fully reduced FMNH-, initially formed via NADH-mediated reduction, to the Fe2S2 cluster. This Fe2S2 cluster is not part of the on-path chain of iron-sulfur clusters connecting the FMN of FdsB with the active-site molybdenum center of FdsA. According to the NADH-bound structure, the nicotinamide ring stacks onto the re-face of the FMN. However, NADH binding significantly reduced the electron density for the isoalloxazine ring of FMN and induced a conformational change in residues of the FMN-binding pocket that display peptide-bond flipping upon NAD+ binding in proper NADH dehydrogenases.

中文翻译:

来自 Cupriavidus necator 的胞质甲酸脱氢酶 FdsABG 的 FdsBG 亚复合体的晶体学和动力学分析。

甲酸氧化成二氧化碳是一碳化合物代谢中的关键反应,其逆反应是许多厌氧生物的产乙酸和产甲烷分支中碳同化的第一步。含钼脱氢酶 FdsABG 是一种可溶性 NAD+ 依赖性甲酸脱氢酶,是 NADH 脱氢酶超家族的成员。在这里,我们展示了来自氢氧化细菌 Cupriavidus necator H16 的胞质 FdsABG 甲酸脱氢酶的 FdsBG 亚复合体的第一个结构,其中包含和不包含结合的 NADH。结构表明,与其他 NADH 脱氢酶相比,FdsB 中的 Fe4S4 和 FdsG 中的 Fe2S2 这两个铁硫簇更接近 FMN。FdsBG 的 NADH 还原的快速冷冻淬火样品的快速动力学研究和 EPR 测量确定了一种中性黄素半醌,FMNH•,以前没有观察到它参与 NADH 介导的 FdsABG 全酶还原。我们发现这种半醌通过将一个电子从完全还原的 FMNH-(最初通过 NADH 介导的还原形成)转移到 Fe2S2 簇而形成。该 Fe2S2 簇不是连接 FdsB 的 FMN 与 FdsA 的活性位点钼中心的铁硫簇路径链的一部分。根据 NADH 结合结构,烟酰胺环堆叠在 FMN 的背面。然而,
更新日期:2020-05-08
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