Understanding the roles of strictly conserved tryptophan residues in O2 producing chlorite dismutases.
Understanding the roles of strictly conserved tryptophan residues in O2 producing chlorite dismutases.
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DOI：
10.1039/c2dt32312e
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发表时间：
2013-03-07
期刊：
影响因子：
--
通讯作者：
DuBois JL
中科院分区：
文献类型：
--
作者：
Blanc B;Rodgers KR;Lukat-Rodgers GS;DuBois JL
在高氯酸盐呼吸细菌中，氯离子歧化酶（Clds）将ClO 2-降解为O2和Cl-，它们在其他不同微生物中的细胞作用仍然不明确。这些蛋白质在血红素的近端侧共享3个高度保守的Trp残基，W155、W156和W227。来自芳香脱氯单胞菌（Dechloromonasaromatica，DaCld）的Cld在与ClO 2 −和过氧乙酸反应时会形成蛋白质基自由基。保守的色氨酸残基在自由基生成和酶功能的作用进行了评估，通过光谱和动力学分析其苯丙氨酸突变体。W155 F突变体受影响最大，似乎失去了WT蛋白的特征性五聚体寡聚化状态、二级结构和血红素结合特性。W156 F突变体最初保留了WT蛋白的许多特征，但随着时间的推移获得了W155 F的许多特征。通过稀释加速转化为无活性的无血红素形式，表明蛋白质的五聚体状态丧失。因此，W155和W156对于血红素结合和蛋白质的反应性五聚体结构的维持都是重要的。相比之下，W227 F保留了WT蛋白的许多特性。在与过氧乙酸（PAA）的瞬态动力学反应中注意到重要的差异，其中W227 F似乎形成含[Fe（IV）=O]的中间体，其随后转化为未偶联的[Fe（IV）=O + AA+]。系统以[PAA]依赖的方式。这与WT蛋白中偶联至卟啉π-阳离子自由基的[Fe（IV）=O]的过氧化物酶样形成相反，其以[PAA]-独立的方式衰变。这些观察结果和缺乏氧化还原保护的血红素在任何色氨酸突变体表明蛋白质自由基形成的趋势，在DaCld是独立的任何这些保守的活性位点残基。
The chlorite dismutases (Clds) degrade ClO2− to O2 and Cl− in perchlorate respiring bacteria, and they serve still poorly defined cellular roles in other diverse microbes. These proteins share 3 highly conserved Trp residues, W155, W156, and W227, on the proximal side of the heme. The Cld from Dechloromonas aromatica (DaCld) has been shown to form protein-based radicals in its reactions with ClO2− and peracetic acid. The roles of the conserved Trp residues in radical generation and in enzymatic function were assessed via spectroscopic and kinetic analysis of their Phe mutants. The W155F mutant was the most dramatically affected, appearing to lose the characteristic pentameric oligomerization state, secondary structure, and heme binding properties of the WT protein. The W156F mutant initially retains many features of the WT protein but over time acquires many of the features of W155F. Conversion to an inactive, heme-free form is accelerated by dilution, suggesting loss of the protein’s pentameric state. Hence, both W155 and W156 are important for heme binding and maintenance of the protein’s reactive pentameric structure. W227F by contrast retains many properties of the WT protein. Important differences are noted in the transient kinetic reactions with peracetic acid (PAA), where W227F appears to form an [Fe(IV)=O]-containing intermediate, which subsequently converts to an uncoupled [Fe(IV)=O + AA+.] system in a [PAA]-dependent manner. This is in contrast to the peroxidase-like formation of [Fe(IV)=O] coupled to a porphyrin π-cation radical in the WT protein, which decays in a [PAA]-independent manner. These observations and the lack of redox protection for the heme in any of the Trp mutants suggests a tendency for protein radical formation in DaCld that is independent of any of these conserved active site residues.
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DOI：
10.1038/nsb1297-1032
发表时间：
1997-12-01
期刊：
NATURE STRUCTURAL BIOLOGY
影响因子：
--
作者：
Gajhede, M;Schuller, DJ;Poulos, TL
通讯作者：
Poulos, TL
影响因子：
3.5
作者：
MITTLER, R;ZILINSKAS, BA
通讯作者：
ZILINSKAS, BA
影响因子：
4.8
作者：
Morimoto, A;Tanaka, M;Morishima, I
通讯作者：
Morishima, I
影响因子：
4.8
作者：
RodriguezLopez, JN;HernandezRuiz, J;Arnao, MB
通讯作者：
Arnao, MB
影响因子：
5.6
作者：
Goblirsch B;Kurker RC;Streit BR;Wilmot CM;DuBois JL
通讯作者：
DuBois JL