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

• Type of Material: Article
• Authors: Sahand Emamian, Emory University; Kendra A Ireland, Emory University; Vatsal Purohit, Emory University; Kirklin L Mcwhorter, Emory University; Olga Maximova, Purdue University; Winter Allen, Purdue University; Scott Jensen, Purdue University; Diego M Casa, The Advanced Photon Source; Yulia Pushkar, Purdue University; Katherine Davis, Emory University
• Language: English
• Date: 2023-01-12
• Publisher: ACS Publicatioms
• Publication Version: Final Published Version
• Copyright Statement: © 2022 American Chemical Society
• License: Creative Commons Attribution-NonCommercial 4.0 International
• Final Published Version (URL): http://dx.doi.org/10.1021/acs.jpclett.2c03018
• Title of Journal or Parent Work: Journal of Physical Chemistry Letters
• Volume: 14
• Issue: 1
• Start Page: 41
• End Page: 48
• Keywords: Metalloproteins; Catalytic Domain; Metals; Heme; Spectrometry, X-Ray Emission

Abstract

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- A nd low-spin states. These experiments validate the suitability of an X-ray emission approach for determining spin states of previously uncharacterized metalloenzyme reaction intermediates.