X‑ray Emission Spectroscopy of Single Protein Crystals Yields Insights into Heme Enzyme Intermediates

Emamian, Sahand; Ireland, Kendra A.; Purohit, Vatsal; McWhorter, Kirklin L.; Maximova, Olga; Allen, Winter; Jensen, Scott; Casa, Diego M.; Pushkar, Yulia; Davis, Katherine M.

doi:10.1021/acs.jpclett.2c03018

Title: X‑ray Emission Spectroscopy of Single Protein Crystals Yields Insights into Heme Enzyme Intermediates

Journal Article · Sun Dec 25 00:00:00 EST 2022 · Journal of Physical Chemistry Letters

DOI:https://doi.org/10.1021/acs.jpclett.2c03018· OSTI ID:2229096

Emamian, Sahand; Ireland, Kendra A.; Purohit, Vatsal; McWhorter, Kirklin L.; Maximova, Olga; Allen, Winter; Jensen, Scott; Casa, Diego M.; Pushkar, Yulia; Davis, Katherine M.

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.

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Research Organization:: Argonne National Laboratory (ANL), Argonne, IL (United States)

Sponsoring Organization:: National Institutes of Health (NIH); National Science Foundation (NSF); Canadian Light Source, Inc.; USDOE Office of Science - Office of Basic Energy Sciences - Scientific User Facilities Division

DOE Contract Number:: AC02-06CH11357

OSTI ID:: 2229096

Journal Information:: Journal of Physical Chemistry Letters, Vol. 14, Issue 1

Country of Publication:: United States

Language:: English

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