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首页> 外文期刊>Applied Microbiology >Purification and Characterization of the [NiFe]-Hydrogenase of Shewanella oneidensis MR-1
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Purification and Characterization of the [NiFe]-Hydrogenase of Shewanella oneidensis MR-1

机译:沙瓦氏假单胞菌MR-1 [NiFe]-加氢酶的纯化和鉴定

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摘要

Shewanella oneidensis MR-1 possesses a periplasmic [NiFe]-hydrogenase (MR-1 [NiFe]-H_(2)ase) that has been implicated in H_(2) production and oxidation as well as technetium [Tc(VII)] reduction. To characterize the roles of MR-1 [NiFe]-H_(2)ase in these proposed reactions, the genes encoding both subunits of MR-1 [NiFe]-H_(2)ase were cloned and then expressed in an MR-1 mutant without hyaB and hydA genes. Expression of recombinant MR-1 [NiFe]-H_(2)ase in trans restored the mutant's ability to produce H_(2) at 37% of that for the wild type. Following purification, MR-1 [NiFe]-H_(2)ase coupled H_(2) oxidation to reduction of Tc(VII)O_(4)~(?) and methyl viologen. Change of the buffers used affected MR-1 [NiFe]-H_(2)ase-mediated reduction of Tc(VII)O_(4)~(?) but not methyl viologen. Under the conditions tested, all Tc(VII)O_(4)~(?) used was reduced in Tris buffer, while in HEPES buffer, only 20% of Tc(VII)O_(4)~(?) was reduced. The reduced products were soluble in Tris buffer but insoluble in HEPES buffer. Transmission electron microscopy analysis revealed that Tc precipitates reduced in HEPES buffer were aggregates of crystallites with diameters of ~5 nm. Measurements with X-ray absorption near-edge spectroscopy revealed that the reduction products were a mixture of Tc(IV) and Tc(V) in Tris buffer but only Tc(IV) in HEPES buffer. Measurements with extended X-ray adsorption fine structure showed that while the Tc bonding environment in Tris buffer could not be determined, the Tc(IV) product in HEPES buffer was very similar to Tc(IV)O_(2)· n H_(2)O, which was also the product of Tc(VII)O_(4)~(?) reduction by MR-1 cells. These results shows for the first time that MR-1 [NiFe]-H_(2)ase catalyzes Tc(VII)O_(4)~(?) reduction directly by coupling to H_(2) oxidation.
机译:拟人希瓦氏菌MR-1具有周质性[NiFe]-氢化酶(MR-1 [NiFe] -H_(2)ase),与H_(2)的生产和氧化以及tech [Tc(VII)]的还原有关。 。为了表征MR-1 [NiFe] -H_(2)ase在这些拟议的反应中的作用,克隆了编码MR-1 [NiFe] -H_(2)ase的两个亚基的基因,然后在MR-1中表达没有hyaB和hydA基因的突变体。重组MR-1 [NiFe] -H_(2)酶的反式表达恢复了突变体产生H_(2)的能力,为野生型的37%。纯化后,MR-1 [NiFe] -H_(2)酶将H_(2)氧化偶联以还原Tc(VII)O_(4)-(α)和甲基紫精。所用缓冲液的变化会影响MR-1 [NiFe] -H_(2)酶介导的Tc(VII)O_(4)〜(α)还原,但不会影响甲基紫精。在测试条件下,所有使用的Tc(VII)O_(4)〜(α)在Tris缓冲液中均被还原,而在HEPES缓冲液中,只有20%的Tc(VII)O_(4)〜(α)被还原。还原产物可溶于Tris缓冲液,但不溶于HEPES缓冲液。透射电子显微镜分析表明,在HEPES缓冲液中还原的Tc沉淀物是直径约5 nm的微晶聚集体。 X射线吸收近边缘光谱法的测量结果表明,还原产物是Tris缓冲液中Tc(IV)和Tc(V)的混合物,而HEPES缓冲液中只有Tc(IV)。 X射线吸附精细结构的扩展测量表明,虽然无法确定Tris缓冲液中的Tc键合环境,但HEPES缓冲液中的Tc(IV)产物与Tc(IV)O_(2)·n H_(2 )O,也是MR-1细胞还原Tc(VII)O_(4)〜(α)的产物。这些结果首次表明,MR-1 [NiFe] -H_(2)酶直接与H_(2)氧化偶联,催化Tc(VII)O_(4)〜(α)还原。

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