Pelzmann Astrid, Ferner Marion, Gnida Manuel, Meyer-Klaucke Wolfram, Maisel Tobias, Meyer Ortwin
University of Bayreuth, Universitätsstrasse 30, Bayreuth 95440, Germany.
J Biol Chem. 2009 Apr 3;284(14):9578-86. doi: 10.1074/jbc.M805354200. Epub 2009 Feb 2.
CO dehydrogenase from the Gram-negative chemolithoautotrophic eubacterium Oligotropha carboxidovorans OM5 is a structurally characterized molybdenum-containing iron-sulfur flavoenzyme, which catalyzes the oxidation of CO (CO + H(2)O --> CO(2) + 2e(-) + 2H(+)). It accommodates in its active site a unique bimetallic [CuSMoO(2)] cluster, which is subject to post-translational maturation. Insertional mutagenesis of coxD has established its requirement for the assembly of the [CuSMoO(2)] cluster. Disruption of coxD led to a phenotype of the corresponding mutant OM5 D::km with the following characteristics: (i) It was impaired in the utilization of CO, whereas the utilization of H(2) plus CO(2) was not affected; (ii) Under appropriate induction conditions bacteria synthesized a fully assembled apo-CO dehydrogenase, which could not oxidize CO; (iii) Apo-CO dehydrogenase contained a [MoO(3)] site in place of the [CuSMoO(2)] cluster; and (iv) Employing sodium sulfide first and then the Cu(I)-(thiourea)(3) complex, the non-catalytic [MoO(3)] site could be reconstituted in vitro to a [CuSMoO(2)] cluster capable of oxidizing CO. Sequence information suggests that CoxD is a MoxR-like AAA+ ATPase chaperone related to the hexameric, ring-shaped BchI component of Mg(2+)-chelatases. Recombinant CoxD, which appeared in Escherichia coli in inclusion bodies, occurs exclusively in cytoplasmic membranes of O. carboxidovorans grown in the presence of CO, and its occurrence coincided with GTPase activity upon sucrose density gradient centrifugation of cell extracts. The presumed function of CoxD is the partial unfolding of apo-CO dehydrogenase to assist in the stepwise introduction of sulfur and copper in the [MoO(3)] center of the enzyme.
来自革兰氏阴性化能无机自养真细菌嗜羧寡养单胞菌OM5的一氧化碳脱氢酶是一种结构已得到表征的含钼铁硫黄素酶，它催化一氧化碳的氧化反应（CO + H₂O → CO₂ + 2e⁻ + 2H⁺）。其活性位点容纳一个独特的双金属[CuSMoO₂]簇，该簇会经历翻译后成熟过程。coxD的插入诱变已确定其对于[CuSMoO₂]簇组装的必要性。coxD的破坏导致相应突变体OM5 D::km具有以下特征的表型：（i）其利用CO的能力受损，而H₂加CO₂的利用不受影响；（ii）在适当的诱导条件下，细菌合成了完全组装好的脱辅基一氧化碳脱氢酶，该酶无法氧化CO；（iii）脱辅基一氧化碳脱氢酶含有一个[MoO₃]位点，取代了[CuSMoO₂]簇；（iv）先使用硫化钠，然后使用Cu(I)-(硫脲)₃络合物，非催化性的[MoO₃]位点可在体外重构成能够氧化CO的[CuSMoO₂]簇。序列信息表明CoxD是一种与Mg²⁺螯合酶的六聚体环状BchI组分相关的MoxR样AAA⁺ ATP酶伴侣蛋白。在大肠杆菌中以包涵体形式出现的重组CoxD仅存在于在CO存在下生长的嗜羧寡养单胞菌的细胞质膜中，并且在对细胞提取物进行蔗糖密度梯度离心时，其出现与GTP酶活性一致。CoxD的推测功能是使脱辅基一氧化碳脱氢酶部分展开，以协助在该酶的[MoO₃]中心逐步引入硫和铜。
