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

05 October 2022, Version 2
This content is a preprint and has not undergone peer review at the time of posting.

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 3rd 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.

Keywords

metalloenzyme
spin
covalency
indoleamine 2
3-dioxygenase
iron
X-ray emission spectroscopy
von Hamos
density functional theory

Supplementary materials

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Title
Supporting Information for X-ray Emission Spectroscopy of Single Protein Crystals Yields Insights into Heme Enzyme Intermediates
Description
Supplementary Materials for "Supporting Information for X-ray Emission Spectroscopy of Single Protein Crystals Yields Insights into Heme Enzyme Intermediates"
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