Structural Properties and Catalytic Implications of the SPASM Domain Iron–Sulfur Clusters in Methylorubrum extorquens PqqE,Journal of the American Chemical Society

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Structural Properties and Catalytic Implications of the SPASM Domain Iron–Sulfur Clusters in Methylorubrum extorquens PqqE
Journal of the American Chemical Society ( IF 14.4 ) Pub Date : 2020-07-09 , DOI: 10.1021/jacs.0c02044
Wen Zhu ₁ , Lindsey M Walker ₂ , Lizhi Tao ₃ , Anthony T Iavarone ₁ , Xuetong Wei ₄ , R David Britt ₃ , Sean J Elliott ₂ , Judith P Klinman _{1,

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Affiliation

Understanding the relationship between the metallocofactor and its protein environment is the key to uncovering the mechanism of metalloenzymes. PqqE, a radical S-adenosylmethionine enzyme in pyrroloquinoline quinone (PQQ) biosynthesis, contains three iron-sulfur cluster binding sites. Two auxiliary iron-sulfur cluster binding sites, designated as AuxI and AuxII, use distinctive ligands compared to other proteins in the family while their functions remain unclear. Here, we investigate the electronic properties of these iron-sulfur clusters and compare the catalytic efficiency of wild-type (WT) Methylorubrum extorquens AM1 PqqE to a range of mutated constructs. Using native mass spectrometry, protein film electrochemistry, and electron paramagnetic resonance spectroscopy, we confirm the previously proposed incorporation of a mixture of [2Fe-2S] and [4Fe-4S] clusters at the AuxI site and are able to assign redox potentials to each of the three iron-sulfur clusters. Significantly, a conservative mutation at AuxI, C268H, shown to selectively incorporate a [4Fe-4S] cluster, catalyzes an enhancement of uncoupled S-adenosylmethionine cleavage relative to WT, together with the elimination of detectable peptide cross-linked product. While a [4Fe-4S] cluster can be tolerated at the AuxI site, the aggregate findings suggest a functional [2Fe-2S] configuration within the AuxI site. PqqE variants with nondestructive ligand replacements at AuxII also show that the reduction potential at this site can be manipulated by changing the electronegativity of the unique aspartate ligand. A number of novel mechanistic features are proposed based on the kinetic and spectroscopic data. Additionally, bioinformatic analyses suggest that the unique ligand environment of PqqE may be relevant to its role in PQQ biosynthesis within an oxygen-dependent biosynthetic pathway.

中文翻译：

Methylorubrum extorquens PqqE中SPASM域铁-硫簇的结构特性和催化意义

了解金属辅因子与其蛋白质环境的关系是揭示金属酶作用机制的关键。PqqE 是吡咯并喹啉醌 (PQQ) 生物合成中的一种自由基 S-腺苷甲硫氨酸酶，包含三个铁硫簇结合位点。两个辅助铁硫簇结合位点，指定为 AuxI 和 AuxII，与家族中的其他蛋白质相比，它们使用独特的配体，但它们的功能尚不清楚。在这里，我们研究了这些铁硫簇的电子特性，并比较了野生型 (WT) Methylorubrum extorquens AM1 PqqE 与一系列突变结构的催化效率。使用原生质谱、蛋白质膜电化学和电子顺磁共振波谱，我们确认了先前提议在 AuxI 位点掺入 [2Fe-2S] 和 [4Fe-4S] 簇的混合物，并且能够为三个铁硫簇中的每一个分配氧化还原电位。重要的是，AuxI 上的保守突变 C268H 显示出选择性掺入 [4Fe-4S] 簇，催化相对于 WT 的未偶联 S-腺苷甲硫氨酸裂解的增强，同时消除可检测的肽交联产物。虽然在 AuxI 站点可以容忍 [4Fe-4S] 簇，但汇总结果表明 AuxI 站点内具有功能性的 [2Fe-2S] 配置。在 AuxII 处具有非破坏性配体替换的 PqqE 变体也表明，可以通过改变独特天冬氨酸配体的电负性来操纵该位点的还原电位。基于动力学和光谱数据提出了许多新的机械特征。此外，生物信息学分析表明，PqqE 独特的配体环境可能与其在氧依赖性生物合成途径中的 PQQ 生物合成中的作用有关。

更新日期：2020-07-09

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