CD and MCD of CytC3 and taurine dioxygenase:: Role of the facial triad in α-KG-dependent oxygenases
CD and MCD of CytC3 and taurine dioxygenase:: Role of the facial triad in α-KG-dependent oxygenases
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DOI：
10.1021/ja074557r
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发表时间：
2007-11-21
影响因子：
15
通讯作者：
Solomon, Edward I.
中科院分区：
文献类型：
--
作者：
Neidig, Michael L.;Brown, Christina D.;Solomon, Edward I.
α-酮戊二酸 (α-KG) 依赖性加氧酶是一大类多样化的单核非血红素铁酶，需要 Fe-II、α-KG 和双氧进行催化，而 α-KG 共底物为氧活化提供额外的还原当量。虽然这些系统表现出各种各样的反应性（即羟基化、去饱和、闭环等），但它们在 Fe-II 活性位点上都有一个共同的结构基序，称为 2-His-1-羧酸酯面部三联体。最近，已鉴定出一种新的 α-KG 依赖性加氧酶亚类，它表现出新颖的反应活性，即未活性碳中心的氧化卤化。这些酶在结构上也很独特，因为它们不包含标准的面部三联体，因为 Cl- 配体代替了羧酸盐进行配位。将涉及 CD、MCD 和 VTVH MCD 光谱的 Fe-II 方法应用于 CytC3，以阐明配位球扰动的活性位点结构效应。观察到 Fell 对 apo-CytC3 的亲和力显着降低，支持面部三联体对于铁协调形成休息位点的必要性。此外，在 Fe-II/α-KG 复合物中观察到的有趣差异相对于其他 α-KG 依赖性加氧酶中的同源复合物，表明存在具有弱水配体的扭曲 6C 位点。结合牛磺酸双加氧酶的平行研究和过去对克拉维酸合酶的研究，这些结果定义了面部三联体的羧酸盐配体通过羧酸盐的非配位氧与配位水之间的氢键相互作用来稳定水配位的作用。这些研究提供了对活性位点特征的初步了解，这些特征有利于 CytC3 的氯化作用，而不是相关酶中发生的羟基化反应。
The a-ketoglutarate (alpha-KG)-dependent oxygenases are a large and diverse class of mononuclear non-heme iron enzymes that require Fe-II, alpha-KG, and dioxygen for catalysis with the alpha-KG cosubstrate supplying the additional reducing equivalents for oxygen activation. While these systems exhibit a diverse array of reactivities (i.e., hydroxylation, desaturation, ring closure, etc.), they all share a common structural motif at the Fe-II active site, termed the 2-His-1-carboxylate facial triad. Recently, a new subclass of alpha-KG-dependent oxygenases has been identified that exhibits novel reactivity, the oxidative halogenation of unactivated carbon centers. These enzymes are also structurally unique in that they do not contain the standard facial triad, as a Cl- ligand is coordinated in place of the carboxylate. An Fe-II methodology involving CD, MCD, and VTVH MCD spectroscopies was applied to CytC3 to elucidate the active-site structural effects of this perturbation of the coordination sphere. A significant decrease in the affinity of Fell for apo-CytC3 was observed, supporting the necessity of the facial triad for iron coordination to form the resting site. In addition, interesting differences observed in the Fe-II/alpha-KG complex relative to the cognate complex in other alpha-KG-dependent oxygenases indicate the presence of a distorted 6C site with a weak water ligand. Combined with parallel studies of taurine dioxygenase and past studies of clavaminate synthase, these results define a role of the carboxylate ligand of the facial triad in stabilizing water coordination via a H-bonding interaction between the noncoordinating oxygen of the carboxylate and the coordinated water. These studies provide initial insight into the active-site features that favor chlorination by CytC3 over the hydroxylation reactions occurring in related enzymes.