Determination of the iron(IV) local spin states of the Q intermediate of soluble methane monooxygenase by Kβ X-ray emission spectroscopy.
Determination of the iron(IV) local spin states of the Q intermediate of soluble methane monooxygenase by Kβ X-ray emission spectroscopy.
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DOI：
10.1007/s00775-022-01953-4
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发表时间：
2022-09
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可溶性甲烷单加氧酶（sMMO）促进甲烷在非血红素FeIV 2中间体MMOHQ处转化为甲醇，所述中间体MMOHQ在催化循环期间在sMMO羟化酶组分（MMOH）的活性位点中形成。其他生物系统也在催化中使用高价FeIV位点;然而，MMOHQ作为自然界唯一确定的FeIV 2中间体是独特的。以前的57 Fe穆斯堡尔谱学研究表明，MMOHQ利用两个FeIV位点的反铁磁耦合产生抗磁性簇。不幸的是，这种净自旋的缺乏阻止了大多数光谱技术对每个铁的局部自旋态（Sloc）的测定。在这里，我们使用Fe Kβ X射线发射光谱（XES）来表征sMMO催化循环的关键中间体的局部自旋状态，包括通过快速冷冻淬火技术捕获的MMOHQ。MMOHQ的纯XES光谱是通过在穆斯堡尔定量的帮助下减去来自其他反应循环中间体的贡献而获得的。比较MMOHQ光谱与已知的Sloc = 1和Sloc = 2铁IV网站在化学和生物学模型揭示，MMOHQ具有Sloc = 2铁网站。局部自旋状态的实验测定将有助于指导未来sMMO催化的计算和机理研究。在线版本包含补充材料，可通过10.1007/s 00775 -022-01953-4获得。
Soluble methane monooxygenase (sMMO) facilitates the conversion of methane to methanol at a non-heme FeIV2 intermediate MMOHQ, which is formed in the active site of the sMMO hydroxylase component (MMOH) during the catalytic cycle. Other biological systems also employ high-valent FeIV sites in catalysis; however, MMOHQ is unique as Nature’s only identified FeIV2 intermediate. Previous 57Fe Mössbauer spectroscopic studies have shown that MMOHQ employs antiferromagnetic coupling of the two FeIV sites to yield a diamagnetic cluster. Unfortunately, this lack of net spin prevents the determination of the local spin state (Sloc) of each of the irons by most spectroscopic techniques. Here, we use Fe Kβ X-ray emission spectroscopy (XES) to characterize the local spin states of the key intermediates of the sMMO catalytic cycle, including MMOHQ trapped by rapid-freeze-quench techniques. A pure XES spectrum of MMOHQ is obtained by subtraction of the contributions from other reaction cycle intermediates with the aid of Mössbauer quantification. Comparisons of the MMOHQ spectrum with those of known Sloc = 1 and Sloc = 2 FeIV sites in chemical and biological models reveal that MMOHQ possesses Sloc = 2 iron sites. This experimental determination of the local spin state will help guide future computational and mechanistic studies of sMMO catalysis. The online version contains supplementary material available at 10.1007/s00775-022-01953-4.
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