ROLE OF CYSTEINE-337 AND CYSTEINE-340 IN FLAVOPROTEIN THAT FUNCTIONS AS NADH OXIDASE FROM AMPHIBACILLUS-XYLANUS STUDIED BY SITE-DIRECTED MUTAGENESIS
ROLE OF CYSTEINE-337 AND CYSTEINE-340 IN FLAVOPROTEIN THAT FUNCTIONS AS NADH OXIDASE FROM AMPHIBACILLUS-XYLANUS STUDIED BY SITE-DIRECTED MUTAGENESIS
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DOI：
10.1074/jbc.270.11.5812
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发表时间：
1995-03-17
影响因子：
4.8
通讯作者：
NISHINO, T
中科院分区：
文献类型：
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作者：
OHNISHI, K;NIIMURA, Y;NISHINO, T
木双胞菌的黄素蛋白催化氧还原为过氧化氢，四聚体中的每个多肽链含有5个半胱氨酸残基，二亚硫基完全还原酶需要6个电子，这种行为表明除了FAD外，还存在氧化还原中心，这些可能是二硫化物。为了评估二硫键在酶中的催化作用，两个半胱氨酸(Cys-337和Cys-340)与烷基过氧化氢还原酶F52a蛋白和硫氧还蛋白还原酶具有高度同源性，通过对克隆的黄素基因进行定点突变(单独和双突变)，将其中两个半胱氨酸(Cys-337和Cys-340)改变为丝氨酸缺少Cys-337和/或Cys-340，只需要2个电子方程就能达到还原黄素态。这些结果表明没有氧化还原活性的二硫键，并证明了Cys-337和Cys-340参与了氧化还原活性的二硫键。通过稳态分析检测了这三种酶的催化活性，对NADH和氧的K-m和K(CAT)值与野生型基本相同，在游离Fad的存在下，NADH氧化酶的活性也显著加速。所有突变体酶的NADH：5，5‘-二硫双(2-硝基苯甲酸)(DTNB)氧化还原酶活性均低于野生型酶活力的3%，缺失Cys-337或Cys-340的突变体酶活性较弱可能是通过FADH(2)直接还原混合二硫键Cys-337-硫醇或Cys-340-硫醇与亚硝基硫代苯甲酸酯所致。然而，在缺乏这两种半胱氨酸的突变酶中，DTNB还原酶的活性很弱，这表明FADH(2)可以直接还原DTNB或另一种二硫键，尽管效率不高，这些结果表明黄素蛋白中存在分子内的二硫醇-二硫键交换反应。
A flavoprotein from Amphibacillus xylanus catalyzes the reduction of oxygen to hydrogen peroxide, Each polypeptide chain in the tetrameric enzyme contains 5 cysteine residues, The complete reduction of enzyme by dithionite requires 6 electrons, Such behavior indicates the presence of redox centers in addition to the FAD, and these could be disulfides, In order to assess the catalytic role of disulfide in the enzyme, 2 of the cysteines (Cys-337 and Cys-340), which show a high degree of homology with alkyl hydroperoxide reductase F52a protein and thioredoxin reductase, have been changed to serines by site-directed mutagenesis of the cloned flavoprotein gene (individually and in a double mutant), Titration of the three mutant enzymes, lacking Cys-337, Cys-340, or both cysteines, requires only 2 electron eq to reach the reduced flavin state, These results indicate the absence of a redox-active disulfide and demonstrate the involvement of Cys-337 and Cys-340 in the redox-active disulfide, The catalytic activity of the three enzymes was examined by steady-state analysis, The K-m for NADH and oxygen and the k(cat) value of these mutant enzymes were essentially the same as those of wild type, The NADH oxidase activities were also accelerated markedly in the presence of free FAD, which is the case for wild-type enzyme, The NADH:5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) oxidoreductase activities of all mutant enzymes were less than 3% of the activity of wild-type enzyme, The weak DTNB reductase activities in the mutant enzymes lacking Cys-337 or Cys-340 may occur through direct reduction of the mixed disulfide Cys-337-thiol or Cys-340-thiol and nitrothiobenzoate by FADH(2). However, the weak DTNB reductase activity in the mutant enzyme lacking both cysteines indicates that FADH(2) can reduce either DTNB or another disulfide directly, albeit inefficiently, These results suggest intramolecular dithiol-disulfide interchange reactions in the flavoprotein.