CHARACTERIZATION OF THE NICKEL-IRON PERIPLASMIC HYDROGENASE FROM DESULFOVIBRIO-FRUCTOSOVORANS
CHARACTERIZATION OF THE NICKEL-IRON PERIPLASMIC HYDROGENASE FROM DESULFOVIBRIO-FRUCTOSOVORANS
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DOI：
10.1111/j.1432-1033.1990.tb15347.x
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发表时间：
1990-02-14
期刊：
影响因子：
--
通讯作者：
CAMMACK, R
中科院分区：
文献类型：
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作者：
HATCHIKIAN, CE;TRAORE, AS;CAMMACK, R
来自在果糖/硫酸盐培养基上生长的脱硫弧菌的周质氢化酶被纯化至同质。它的分子量为 88 kDa，由 60 kDa 和 28.5 kDa 两个不同的亚基组成。该酶的吸收光谱是铁硫蛋白的特征，其在400和280 nm处的吸收系数分别为50和180 mM-1 cm-1。 D. fructosovorans 氢化酶含有 11 个.+-。 1个铁原子，0.9.+-。 0.15个镍原子和12个.+-。 1 个对酸不稳定的硫原子/分子，但不含硒。该蛋白质及其亚基的氨基酸组成，以及小亚基和大亚基的 N 端序列均已确定。蛋白质的半胱氨酸残基分布在大亚基（9 个残基）和小亚基（11 个残基）之间。该酶的电子自旋共振 (ESR) 特性与镍 (III)、[3Fe-4S] 和 [4Fe-4S] 簇的存在一致。在需氧条件下分离的 D. fructosovorans 的氢化酶需要与氢气或其他还原剂一起孵育，以表达其完整的催化活性。在还原条件下孵育 3 小时后，H2 吸收和 H2 释放活性加倍。与来自 D. gigas 的 (NiFe) 氢化酶的比较表明两种蛋白质之间具有很大的结构相似性。然而，两种酶的催化特性之间存在显着差异，这可能与其镍原子各自的状态有关。 ESR 显示 Ni-B 物质的比例较高（g = 2.33、2.16、2.01），这可能与更容易转换为就绪状态有关。讨论了该酶的周质位置和其他细胞区室中氢化酶活性的存在与 D. fructosovorans 积极参与种间氢转移的能力。
The periplasmic hydrogenase from Desulfovibrio fructosovorans grown on fructose/sulfate medium was purified to homogeneity. It exhibits a molecular mass of 88 kDa and is composed of two different subunits of 60 kDa and 28.5 kDa. The absorption spectrum of the enzyme is characteristic of an iron-sulfur protein and its absorption coefficients at 400 and 280 nm are 50 and 180 mM-1 cm-1, respectively. D. fructosovorans hydrogenase contains 11 .+-. 1 iron atoms, 0.9 .+-. 0.15 nickel atom and 12 .+-. 1 acid-labile sulfur atoms/molecule but does not contain selenium. The amino acid composition of the protein and of its subunits, as well as the N-terminal sequences of the small and large subunits, have been determined. The cysteine residues of the protein are distributed between the large (9 residues) and the small subunits (11 residues). Electron spin resonance (ESR) properties of the enzyme are consistent with the presence of nickel (III), [3Fe-4S] and [4Fe-4S] clusters. The hydrogenase of D. fructosovorans isolated under aerobic conditions required an incubation with hydrogen or other reductants in order to express its full catalytic activity. H2 uptake and H2 evolution activities doubled after a 3-h incubation under reducing conditions. Comparison with the (NiFe) hydrogenase from D. gigas shows great structural similarities between the two proteins. However, there are significant differences between the catalytic properties of the two enzymes which can be related to the respective state of their nickel atom. ESR showed a higher proportion of the Ni-B species (g = 2.33, 2.16, 2.01) which can be related to a more facile conversion to the ready state. The periplasmic location of the enzyme and the presence of hydrogenase activity in other cellular compartments are discussed in relation to the ability of D. fructosovorans to participate actively in interspecies hydrogen transfer.