X-ray Emission Spectroscopy of Single Protein Crystals Yields Insights into Heme Enzyme Intermediates
X-ray Emission Spectroscopy of Single Protein Crystals Yields Insights into Heme Enzyme Intermediates
复制标题
DOI：
10.1021/acs.jpclett.2c03018
复制
发表时间：
2022-12-25
影响因子：
5.7
通讯作者：
Davis，Katherine M.
中科院分区：
文献类型：
--
作者：
Emamian，Sahand;Ireland，Kendra A.;Davis，Katherine M.
相关金属辅因子的氧化态、自旋态和金属-配体共价性的变化通常会增强酶的反应性。同时研究这些过程和结构重排的光谱方法的发展对于阐明金属酶的机制是至关重要的。在这里，我们论证了在第三代同步辐射光源上收集金属酶晶体的X射线发射光谱的可行性。特别是，我们报告了von Hamos光谱仪的发展，该光谱仪用于收集Fe-K-β发射，优化了对稀薄生物样品的分析。我们进一步展示了它在免疫抑制的血红素依赖酶吲哚胺2，3-双加氧酶晶体中的应用。将不同状态的蛋白质晶体的光谱与相关的参考化合物进行了比较。评估共价性的互补密度泛函计算支持我们的光谱分析，并确定了与高自旋态和低自旋态相关的活性中心构象。这些实验验证了X射线发射方法用于确定以前未表征的金属酶反应中间体的自旋状态的适用性。
Enzyme reactivity is often enhanced by changes in oxidation state, spin state, and metal–ligand covalency of associated metallocofactors. The development of spectroscopic methods for studying these processes coincidentally with structural rearrangements is essential for elucidating metalloenzyme mechanisms. Herein, we demonstrate the feasibility of collecting X-ray emission spectra of metalloenzyme crystals at a third-generation synchrotron source. In particular, we report the development of a von Hamos spectrometer for the collection of Fe Kβ emission optimized for analysis of dilute biological samples. We further showcase its application in crystals of the immunosuppressive heme-dependent enzyme indoleamine 2,3-dioxygenase. Spectra from protein crystals in different states were compared with relevant reference compounds. Complementary density functional calculations assessing covalency support our spectroscopic analysis and identify active site conformations that correlate to high- and low-spin states. These experiments validate the suitability of an X-ray emission approach for determining spin states of previously uncharacterized metalloenzyme reaction intermediates.