Histidine ligand protonation and redox potential in the Rieske dioxygenases: Role of a conserved aspartate in anthranilate 1,2-dioxygenase
Histidine ligand protonation and redox potential in the Rieske dioxygenases: Role of a conserved aspartate in anthranilate 1,2-dioxygenase
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DOI：
10.1021/bi035385n
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发表时间：
2003-11-25
期刊：
影响因子：
2.9
通讯作者：
Kurtz, DM
中科院分区：
文献类型：
--
作者：
Beharry, ZM;Eby, DM;Kurtz, DM
Rieske 双加氧酶，邻氨基苯甲酸 1,2-双加氧酶，催化邻氨基苯甲酸（2-氨基苯甲酸）的 1,2-二羟基化。与所有特征化的 Rieske 双加氧酶一样，催化转化为二醇发生在双加氧酶组分 AntAB 内的单核铁位点，该位点接受来自近端 Rieske [2Fe-2S] 中心的电子。在相关的萘双加氧酶 (NDO) 中，保守的天冬氨酸残基位于单核中心和 Rieske 铁中心之间，并与 Rieske 中心的组氨酸配体形成氢键。这种天冬氨酸残基的工程取代导致完全失活，这被认为是由于消除了有效的位点间电子转移途径而引起的[Parales, R. E., Parales, J. V., 和 Gibson, D. T. (1999) J. Bacteriol. 181, 1831-1837]。在 AntAB 中用丙氨酸、天冬酰胺或谷氨酸取代相应的天冬氨酸 D218 也会导致酶在很宽的 pH 范围内完全失活，尽管保留了六聚体四级结构和铁中心占据。在中性 pH 条件下，该变体的 Rieske 中心还原电位被测量为比野生型酶负 100 mV。野生型 AntAB 在 pH 9 时完全失活，并表现出改变的 Rieske 中心吸收光谱，类似于中性 pH 下的 D218 变体。这些结果支持该天冬氨酸在维持 Rieske 中心的质子化状态和还原潜力中的作用。野生型和 D218A 变体 AntAB 在停流时间过程中均表现出底物依赖性 Rieske 中心氧化的快速阶段。该观察结果不支持该天冬氨酸在轻松的位点间电子转移途径或 Rieske 中心还原电位的生产性底物门控中的作用。然而，由于单一转换导致野生型酶而非 D218A 变体导致邻氨基苯甲酸二羟基化，因此这种天冬氨酸也必须在单核铁位点处或附近的底物二羟基化中发挥关键作用。
The Rieske dioxygenase, anthranilate 1,2-dioxygenase, catalyzes the 1,2-dihydroxylation of anthranilate (2-aminobenzoate). As in all characterized Rieske dioxygenases, the catalytic conversion to the diol occurs within the dioxygenase component, AntAB, at a mononuclear iron site which accepts electrons from a proximal Rieske [2Fe-2S] center. In the related naphthalene dioxygenase (NDO), a conserved aspartate residue lies between the mononuclear and Rieske iron centers, and is hydrogen-bonded to a histidine ligand of the Rieske center. Engineered substitutions of this aspartate residue led to complete inactivation, which was proposed to arise from elimination of a productive intersite electron transfer pathway [Parales, R. E., Parales, J. V., and Gibson, D. T. (1999) J. Bacteriol. 181, 1831-1837]. Substitutions of the corresponding aspartate, D218, in AntAB with alanine, asparagine, or glutamate also resulted in enzymes that were completely inactive over a wide pH range despite retention of the hexameric quaternary structure and iron center occupancy. The Rieske center reduction potential of this variant was measured to be similar to100 mV more negative than that for the wild-type enzyme at neutral pH. The wild-type AntAB became completely inactive at pH 9 and exhibited an altered Rieske center absorption spectrum which resembled that of the D218 variants at neutral pH. These results support a role for this aspartate in maintaining the protonated state and reduction potential of the Rieske center. Both the wild-type and D218A variant AntABs exhibited substrate-dependent rapid phases of Rieske center oxidations in stopped-flow time courses. This observation does not support a role for this aspartate in a facile intersite electron transfer pathway or in productive substrate gating of the Rieske center reduction potential. However, since the single turnovers resulted in anthranilate dihydroxylation by the wild-type enzyme but not by the D218A variant, this aspartate must also play a crucial role in substrate dihydroxylation at or near the mononuclear iron site.