Max Planck Institute for Chemical Energy Conversion, Stiftstrasse 34-36, 45470, Mülheim an der Ruhr, Germany.
Institute of Inorganic Chemistry, University of Duisburg-Essen, Universitätsstrasse 5-7, 45117, Essen, Germany.
J Biol Inorg Chem. 2022 Sep;27(6):573-582. doi: 10.1007/s00775-022-01953-4. Epub 2022 Aug 21.
Soluble methane monooxygenase (sMMO) facilitates the conversion of methane to methanol at a non-heme Fe intermediate MMOH, which is formed in the active site of the sMMO hydroxylase component (MMOH) during the catalytic cycle. Other biological systems also employ high-valent Fe sites in catalysis; however, MMOH is unique as Nature's only identified Fe intermediate. Previous Fe Mössbauer spectroscopic studies have shown that MMOH employs antiferromagnetic coupling of the two Fe sites to yield a diamagnetic cluster. Unfortunately, this lack of net spin prevents the determination of the local spin state (S) 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 MMOH trapped by rapid-freeze-quench techniques. A pure XES spectrum of MMOH is obtained by subtraction of the contributions from other reaction cycle intermediates with the aid of Mössbauer quantification. Comparisons of the MMOH spectrum with those of known S = 1 and S = 2 Fe sites in chemical and biological models reveal that MMOH possesses S = 2 iron sites. This experimental determination of the local spin state will help guide future computational and mechanistic studies of sMMO catalysis.
可溶性甲烷单加氧酶 (sMMO) 在非血红素 Fe 中间体 MMOH 的作用下促进甲烷向甲醇的转化，该中间体 MMOH 在 sMMO 羟化酶组分 (MMOH) 的活性位点形成，在催化循环过程中。其他生物系统也在催化中使用高价 Fe 位点；然而，MMOH 是自然界中唯一鉴定出的 Fe 中间体。以前的 Fe Mössbauer 光谱研究表明，MMOH 采用两个 Fe 位点的反铁磁耦合来产生抗磁性团簇。不幸的是，这种缺乏净自旋阻止了大多数光谱技术确定每个铁的局部自旋状态 (S)。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 MMOH。通过 Mössbauer 量化来减去其他反应循环中间体的贡献，可以获得 MMOH 的纯 XES 光谱。将 MMOH 光谱与化学和生物模型中已知的 S = 1 和 S = 2 Fe 位点的光谱进行比较表明，MMOH 具有 S = 2 铁位点。这种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。
