pH-dependent structures of the manganese binding sites in oxalate decarboxylase as revealed by high-field electron paramagnetic resonance.
pH-dependent structures of the manganese binding sites in oxalate decarboxylase as revealed by high-field electron paramagnetic resonance.
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高场电子顺磁共振揭示草酸脱羧酶中锰结合位点的 pH 依赖性结构。
DOI：
10.1021/jp9021807
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发表时间：
2009
期刊：
影响因子：
--
通讯作者：
Un,Sun
中科院分区：
文献类型：
--
作者：
Tabares,LeandroC;Gätjens,Jessica;Hureau,Christelle;Burrell,MatthewR;Bowater,Laura;Pecoraro,VincentL;Bornemann,Stephen;Un,Sun
报道了草酸脱羧酶 (OxdC) 的高场电子顺磁共振 (HFEPR) 研究。 OxdC 将草酸盐分解为二氧化碳和甲酸，并拥有两个不同的锰 (II) 结合位点，称为位点−1 和 -2。 Mn(II) 零场相互作用用于探测金属离子的电子态并检查每个 Mn(II) 中心的化学/机械作用。利用高磁场不仅可以解析这两个位点，还可以精确测量每个位点的 Mn(II) 零场参数。光谱表现出令人惊讶的复杂行为作为 pH 的函数。根据零场相互作用识别出六种不同的物种，其中两种对应于站点 1，四种状态对应于站点 2。使用仅影响位点 1 的突变体验证了分配。从位点 -2 的 HFEPR 光谱确定的形态数据与简单的三元平衡模型一致，而位点 1 的 pH 依赖性可以通过单个 pKa 来描述。这种 pH 依赖性与 His 标签的存在无关，也与制剂中每个亚基是否含有 1.2 或 1.6 Mn 无关。根据模型复合物的光谱数据提出了这六个物种的可能结构，并讨论了在 pH 8 下获得的现有蛋白质晶体结构。虽然位点 1 已被确定为活性位点，并且尚未指定位点 2 的作用，但后者的电子结构及其 pH 行为的显着变化（也与该酶的 pH 依赖性活性相匹配）表明，即使草酸向甲酸的转化是在位点 1 进行，位点 2 也可能发挥催化相关作用。
A high-field electron paramagnetic resonance (HFEPR) study of oxalate decarboxylase (OxdC) is reported. OxdC breaks down oxalate to carbon dioxide and formate and possesses two distinct manganese(II) binding sites, referred to as site−1 and −2. The Mn(II) zero-field interaction was used to probe the electronic state of the metal ion and to examine chemical/mechanistic roles of each of the Mn(II) centers. High magnetic-fields were exploited not only to resolve the two sites, but also to measure accurately the Mn(II) zero-field parameters of each of the sites. The spectra exhibited surprisingly complex behavior as a function of pH. Six different species were identified based on their zero-field interactions, two corresponding to site-1 and four states to site-2. The assignments were verified using a mutant that only affected site-1. The speciation data determined from the HFEPR spectra for site −2 was consistent with a simple triprotic equilibrium model, while the pH dependence of site-1 could be described by a single pKa. This pH dependence was independent of the presence of the His-tag and of whether the preparations contained 1.2 or 1.6 Mn per subunit. Possible structures of the six species are proposed based on spectroscopic data from model complexes and existing protein crystallographic structures obtained at pH 8 are discussed. Although site-1 has been identified as the active site and no role has been assigned to site-2, the pronounced changes in the electronic structure of the latter and its pH behavior, which also matches the pH-dependent activity of this enzyme, suggests that even if the conversion of oxalate to formate is carried out at site-1, site-2 likely plays a catalytically relevant role.