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首页> 外文期刊>Biochemistry >Characterization of a tungsten-substituted nitrogenase isolated from Rhodobacter capsulatus.
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Characterization of a tungsten-substituted nitrogenase isolated from Rhodobacter capsulatus.

机译：从荚膜红细菌分离的钨取代的固氮酶的表征。

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In the phototrophic non-sulfur bacterium Rhodobacter capsulatus, the biosynthesis of the conventional Mo-nitrogenase is strictly Mo-regulated. Significant amounts of both dinitrogenase and dinitrogenase reductase were only formed when the growth medium was supplemented with molybdate (1 microM). During cell growth under Mo-deficient conditions, tungstate, at high concentrations (1 mM), was capable of partially (approximately 25%) substituting for molybdate in the induction of nitrogenase synthesis. On the basis of such conditions, a tungsten-substituted nitrogenase was isolated from R. capsulatus with the aid of anfA (Fe-only nitrogenase defective) mutant cells and partially purified by Q-sepharose chromatography. Metal analyses revealed the protein to contain an average of 1 W-, 16 Fe-, and less than 0.01 Mo atoms per alpha(2)beta(2)-tetramer. The tungsten-substituted (WFe) protein was inactive in reducing N(2) and marginally active in acetylene reduction, but it was found to show considerable activity with respect to the generation of H(2) from protons. The EPR spectrum of the WFe protein, recorded at 4 K, exhibited three distinct signals: (i) an S = 3/2 signal, which dominates the low-field region of the spectrum (g = 4.19, 3.93) and is indicative of a tungsten-substituted cofactor (termed FeWco), (ii) a marginal S = 3/2 signal (g = 4.29, 3.67) that can be attributed to residual amounts of FeMoco present in the protein, and (iii) a broad S = 1/2 signal (g = 2.09, 1.95, 1.86) arising from at least two paramagnetic species. Redox titrational analysis of the WFe protein revealed the midpoint potential of the FeWco (E(m) < -200 mV) to be shifted to distinctly lower potentials as compared to that of the FeMoco (E(m) approximately -50 mV) present in the native enzyme. The P clusters of both the WFe and the MoFe protein appear indistinguishable with respect to their midpoint potentials. EPR spectra recorded with the WFe protein under turnover conditions exhibited a 20% decrease in the intensity of the FeWco signal, indicating that the cofactor can be enzymatically reduced only to a small extent. The data presented in the current study demonstrate the pivotal role of molybdenum in optimal N(2) fixation and provides direct evidence that the inability of a tungsten-substituted nitrogenase to reduce N(2) is due to the difficulty to effectively reduce the FeW cofactor beyond its semi-reduced state.
机译：在光养性非硫细菌荚膜红细菌中，常规钼氮酶的生物合成受到严格的钼调节。仅当生长培养基中添加了钼酸盐（1 microM）时，才会形成大量的二氧化氮酶和二氧化氮还原酶。在Mo缺乏条件下的细胞生长过程中，高浓度（1 mM）的钨酸盐能够部分（约25％）取代钼酸盐诱导硝化酶合成。在这种条件的基础上，借助于anfA（仅Fe的固氮酶缺陷）突变细胞从荚膜红球菌中分离出钨取代的固氮酶，并通过Q-琼脂糖色谱法部分纯化。金属分析显示该蛋白质平均每个alpha（2）beta（2）-四聚体平均含有1 W-，16 Fe-和少于0.01 Mo原子。钨取代的（WFe）蛋白在还原N（2）方面无活性，而在乙炔还原方面则略有活性，但是发现从质子生成H（2）方面显示出相当大的活​​性。在4 K处记录的WFe蛋白的EPR光谱显示三个不同的信号：（i）S = 3/2信号，该信号主导光谱的低场区域（g = 4.19，3.93），表明钨取代的辅因子（称为FeWco），（ii）边缘S = 3/2信号（g = 4.29，3.67），可归因于蛋白质中FeMoco的残留量，以及（iii）宽S =至少两个顺磁性物质产生的1/2信号（g = 2.09、1.95、1.86）。 WFe蛋白的氧化还原滴定分析显示，FeWco（E（m）<-200 mV）的中点电势与存在于FeMoco（E（m）约-50 mV）的电势相比明显降低。天然酶。 WFe和MoFe蛋白的P簇在中点电位方面没有区别。在周转条件下用WFe蛋白记录的EPR谱图显示FeWco信号强度降低了20％，表明辅酶只能在很小的程度上被酶还原。当前研究中提供的数据证明了钼在最佳N（2）固定中的关键作用，并提供直接证据表明钨取代的固氮酶无法还原N（2）是由于难以有效降低FeW辅因子超越其半还原状态。

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