Mechanism of sulfide-quinone reductase investigated using site-directed mutagenesis and sulfur analysis
Mechanism of sulfide-quinone reductase investigated using site-directed mutagenesis and sulfur analysis
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DOI：
10.1021/bi026032b
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发表时间：
2002-10-01
期刊：
影响因子：
2.9
通讯作者：
Hauska, G
中科院分区：
文献类型：
--
作者：
Griesbeck, C;Schütz, M;Hauska, G
生物硫化物氧化是发生在所有三个生命领域的反应。在许多细菌中负责该反应的一种酶已被鉴定为硫化物：醌氧化还原酶（SQR）。来自荚膜红细菌的酶是一种外周膜结合的黄素蛋白，分子量约为48 kDa，推测为同源二聚体。在这项工作中，从Rb. capsulatus已经用N-末端His标签修饰，并在大肠杆菌中异源表达和纯化。三个半胱氨酸残基已被证明是必不可少的还原半反应的定点诱变。在每个半胱氨酸突变为丝氨酸后，催化活性几乎完全消失。对于任何突变的酶，都没有观察到在野生型酶中观察到的硫化物还原时荧光的降低。保守的缬氨酸残基突变为天冬氨酸内的第三黄素结合结构域导致大幅降低的底物亲和力，硫化物和醌。两个保守的组氨酸残基已分别突变为丙氨酸。这两种酶都表现出SQR反应的pH依赖性的变化。多硫化物已被确定为一个主要的反应产物，使用光谱和色谱方法。在这些数据的基础上，硫化物依赖的还原和醌依赖的氧化的酶和多硫化物的形成的反应机制提出。
Biological sulfide oxidation is a reaction occurring in all three domains of life. One enzyme responsible for this reaction in many bacteria has been identified as sulfide: quinone oxidoreductase (SQR). The enzyme from Rhodobacter capsulatus is a peripherically membrane-bound flavoprotein with a molecular mass of approximately 48 kDa, presumably acting as a homodimer. In this work, SQR from Rb. capsulatus has been modified with an N-terminal His tag and heterologously expressed in and purified from Escherichia coli. Three cysteine residues have been shown to be essential for the reductive half-reaction by site-directed mutagenesis. The catalytic activity has been nearly completely abolished after mutation of each of the cysteines to serine. A decrease in fluorescence on reduction by sulfide as observed for the wild-type enzyme has not been observed for any of the mutated enzymes. Mutation of a conserved valine residue to aspartate within the third flavin-binding domain led to a drastically reduced substrate affinity, for both sulfide and quinone. Two conserved histidine residues have been mutated individually to alanine. Both of the resulting enzymes exhibited a shift in the pH dependence of the SQR reaction. Polysulfide has been identified as a primary reaction product using spectroscopic and chromatographic methods. On the basis of these data, reaction mechanisms for sulfide-dependent reduction and quinone-dependent oxidation of the enzyme and for the formation of polysulfide are proposed.