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从氧化亚铁硫杆菌中分离的 NAD 还原型[NiFe]-氢化酶脱偶联酶片段的催化特性。

Catalytic properties of the isolated diaphorase fragment of the NAD-reducing [NiFe]-hydrogenase from Ralstonia eutropha.

机构信息

Institute of Biology, Department of Microbiology, Humboldt-Universität zu Berlin, Berlin, Germany.

出版信息

PLoS One. 2011;6(10):e25939. doi: 10.1371/journal.pone.0025939. Epub 2011 Oct 10.

Abstract

The NAD+-reducing soluble hydrogenase (SH) from Ralstonia eutropha H16 catalyzes the H₂-driven reduction of NAD+, as well as reverse electron transfer from NADH to H+, in the presence of O₂. It comprises six subunits, HoxHYFUI₂, and incorporates a [NiFe] H+/H₂ cycling catalytic centre, two non-covalently bound flavin mononucleotide (FMN) groups and an iron-sulfur cluster relay for electron transfer. This study provides the first characterization of the diaphorase sub-complex made up of HoxF and HoxU. Sequence comparisons with the closely related peripheral subunits of Complex I in combination with UV/Vis spectroscopy and the quantification of the metal and FMN content revealed that HoxFU accommodates a [2Fe2S] cluster, FMN and a series of [4Fe4S] clusters. Protein film electrochemistry (PFE) experiments show clear electrocatalytic activity for both NAD+ reduction and NADH oxidation with minimal overpotential relative to the potential of the NAD+/NADH couple. Michaelis-Menten constants of 56 µM and 197 µM were determined for NADH and NAD+, respectively. Catalysis in both directions is product inhibited with K(I) values of around 0.2 mM. In PFE experiments, the electrocatalytic current was unaffected by O₂, however in aerobic solution assays, a moderate superoxide production rate of 54 nmol per mg of protein was observed, meaning that the formation of reactive oxygen species (ROS) observed for the native SH can be attributed mainly to HoxFU. The results are discussed in terms of their implications for aerobic functioning of the SH and possible control mechanism for the direction of catalysis.

摘要

来自恶臭假单胞菌 H16 的 NAD+-还原型可溶性氢化酶 (SH) 在存在 O₂ 的情况下能够催化 NAD+的 H₂驱动还原,以及 NADH 到 H+的反向电子转移。它由六个亚基 HoxHYFUI₂组成,并包含一个 [NiFe] H+/H₂循环催化中心、两个非共价结合的黄素单核苷酸 (FMN) 基团和一个铁硫簇接力用于电子转移。本研究首次对由 HoxF 和 HoxU 组成的脱氨酶亚基复合物进行了表征。与紧密相关的复合物 I 外围亚基的序列比较结合紫外/可见光谱和金属及 FMN 含量的定量分析表明,HoxFU 容纳了一个 [2Fe2S] 簇、FMN 和一系列 [4Fe4S] 簇。蛋白膜电化学 (PFE) 实验显示,相对于 NAD+/NADH 对的电位,NAD+还原和 NADH 氧化都具有明显的电催化活性,过电势最小。分别确定了 NADH 和 NAD+的米氏常数为 56 µM 和 197 µM。两个方向的催化均受产物抑制,K(I) 值约为 0.2 mM。在 PFE 实验中,电催化电流不受 O₂ 的影响,但是在有氧溶液测定中,观察到中等的超氧化物产生速率为 54 nmol 每毫克蛋白,这意味着观察到的天然 SH 形成的活性氧物种 (ROS) 主要归因于 HoxFU。结果从对 SH 的需氧功能的影响及其对催化方向的可能控制机制的角度进行了讨论。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/ed5e/3189943/f59c5b44b6c2/pone.0025939.g001.jpg

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