Structural Properties and Catalytic Implications of the SPASM Domain Iron-Sulfur Clusters in Methylorubrum extorquens PqqE.
Structural Properties and Catalytic Implications of the SPASM Domain Iron-Sulfur Clusters in Methylorubrum extorquens PqqE.
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DOI：
10.1021/jacs.0c02044
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发表时间：
2020-07
影响因子：
15
通讯作者：
Wen Zhu;Lindsey M. Walker;Lizhi Tao;A. Iavarone;Xuetong Wei;R. Britt;S. Elliott;J. Klinman;J. Klinman
中科院分区：
文献类型：
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作者：
Wen Zhu;Lindsey M. Walker;Lizhi Tao;A. Iavarone;Xuetong Wei;R. Britt;S. Elliott;J. Klinman;J. Klinman
了解金属辅助因子与其蛋白环境的关系是揭示金属酶作用机制的关键。PqqE是吡罗喹啉醌（PQQ）生物合成中的一种s -腺苷基蛋氨酸酶，含有3个铁硫簇结合位点。两个辅助铁硫簇结合位点，称为AuxI和AuxII，与家族中的其他蛋白质相比，使用独特的配体，但其功能尚不清楚。在这里，我们研究了这些铁硫簇的电子性质，并比较了野生型（WT） Methylorubrum敲诈勒索AM1 PqqE与一系列突变构建物的催化效率。利用天然质谱、蛋白质膜电化学和电子顺磁共振谱，我们证实了之前提出的在AuxI位点上加入了[2Fe-2S]和[4Fe-4S]簇的混合物，并能够确定三个铁硫簇各自的氧化还原电位。值得注意的是，AuxI上的一个保守突变C268H选择性地结合了一个[4Fe-4S]簇，与WT相比，它催化了不偶联的s-腺苷蛋氨酸裂解的增强，同时消除了可检测到的肽交联产物。虽然在AuxI位点可以容忍[4Fe-4S]簇，但总体结果表明AuxI位点内存在功能性的[2Fe-2S]结构。在AuxII位点具有非破坏性配体替换的PqqE变异也表明，该位点的还原电位可以通过改变独特的天冬氨酸配体的电负性来控制。根据动力学和光谱数据，提出了一些新的机理特征。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PQQ生物合成中的作用有关。
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.