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首页> 美国卫生研究院文献>Biochemical Journal >Purification and properties of the membrane-bound by hydrogenase from Desulfovibrio desulfuricans.
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Purification and properties of the membrane-bound by hydrogenase from Desulfovibrio desulfuricans.

机译：脱硫脱硫弧菌中加氢酶结合的膜的纯化和性质。

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The membrane-bound hydrogenase from the anaerobic sulphate-reducing bacterium Desulfovibrio desulfuricans (Norway strain) has been purified to homogeneity, with an overall 80-fold purification and a specific activity of 70 mumol of H2 evolved/min per mg of protein. The hydrogenase had a relative molecular mass of 58 000 as determined by gel filtration and was estimated to contain six iron atoms and six acid-labile sulphur groups per molecule. The absorption spectrum of the enzyme was characteristic of an iron-sulphur protein. The E400 and E280 were 28 500 and 109 000 M-1.cm-1 respectively. The e.s.r. of the oxidized protein indicated the presence of [4Fe-4S]3+ or [3Fe-3S]3+, and another paramagnetic centre, probably Ni(III). The hydrogenase was inhibited by heavy-metal salts, carbon monoxide and high ionic strength. However, it was resistant to inhibition by thiol-blocking and metal-complexing reagents. N-Bromosuccinimide totally inhibited the enzyme activity at low concentrations. The enzyme was stable to O2 over long periods and to high temperatures. It catalyses both H2-evolution and H2-uptake with a variety of artificial electron carriers. D. desulfuricans cytochrome C3, its natural electron carrier, had a high affinity for the enzyme (Km = 2 microns). Rate enhancement was observed when cytochrome C3 was added to Methyl Viologen in the H2-evolution assay. The pH optimum for H2-evolution was 6.5.
机译：来自厌氧硫酸盐还原细菌Desulfovibrio desulfuricans（Norway菌株）的膜结合氢化酶已纯化至均一，整体纯化80倍，每毫克蛋白质每分钟释放出70摩尔M2 H2。通过凝胶过滤测定，该氢化酶的相对分子量为58 000，并且估计每个分子含有六个铁原子和六个酸不稳定的硫基。该酶的吸收光谱是铁硫蛋白的特征。 E400和E280分别为28500和109 000 M-1.cm-1。 e.s.r.氧化蛋白质的“α”表明存在[4Fe-4S] 3+或[3Fe-3S] 3+，以及另一个顺磁中心，可能是Ni（III）。氢化酶被重金属盐，一氧化碳和高离子强度抑制。但是，它对硫醇封闭剂和金属络合剂的抑制作用有抵抗力。在低浓度时，N-溴代琥珀酰亚胺完全抑制酶的活性。该酶在长期和高温下对氧气稳定。它通过多种人造电子载体催化H2的进化和H2的吸收。 D.desulfuricans细胞色素C3，其天然电子载体，对酶具有很高的亲和力（Km = 2微米）。当在H2进化分析中将细胞色素C3添加到甲基紫精中时，观察到速率提高。 H 2释放的最适pH为6.5。

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