Issue 45, 2022

Capture of activated dioxygen intermediates at the copper-active site of a lytic polysaccharide monooxygenase

Abstract

Metalloproteins perform a diverse array of redox-related reactions facilitated by the increased chemical functionality afforded by their metallocofactors. Lytic polysaccharide monooxygenases (LPMOs) are a class of copper-dependent enzymes that are responsible for the breakdown of recalcitrant polysaccharides via oxidative cleavage at the glycosidic bond. The activated copper-oxygen intermediates and their mechanism of formation remains to be established. Neutron protein crystallography which permits direct visualization of protonation states was used to investigate the initial steps of oxygen activation directly following active site copper reduction in Neurospora crassa LPMO9D. Herein, we cryo-trap an activated dioxygen intermediate in a mixture of superoxo and hydroperoxo states, and we identify the conserved second coordination shell residue His157 as the proton donor. Density functional theory calculations indicate that both superoxo and hydroperoxo active site states are stable. The hydroperoxo formed is potentially an early LPMO catalytic reaction intermediate or the first step in the mechanism of hydrogen peroxide formation in the absence of substrate. We observe that the N-terminal amino group of the copper coordinating His1 remains doubly protonated directly following molecular oxygen reduction by copper. Aided by molecular dynamics and mining minima free energy calculations we establish that the conserved second-shell His161 in MtPMO3* displays conformational flexibility in solution and that this flexibility is also observed, though to a lesser extent, in His157 of NcLPMO9D. The imidazolate form of His157 observed in our structure following oxygen intermediate protonation can be attributed to abolished His157 flexibility due steric hindrance in the crystal as well as the solvent-occluded active site environment due to crystal packing. A neutron crystal structure of NcLPMO9D at low pH further supports occlusion of the active site since His157 remains singly protonated even at acidic conditions.

Graphical abstract: Capture of activated dioxygen intermediates at the copper-active site of a lytic polysaccharide monooxygenase

Supplementary files

Article information

Article type
Edge Article
Submitted
08 Sept. 2022
Accepted
19 Okt. 2022
First published
02 Nov. 2022
This article is Open Access

All publication charges for this article have been paid for by the Royal Society of Chemistry
Creative Commons BY license

Chem. Sci., 2022,13, 13303-13320

Capture of activated dioxygen intermediates at the copper-active site of a lytic polysaccharide monooxygenase

G. C. Schröder, W. B. O'Dell, S. P. Webb, P. K. Agarwal and F. Meilleur, Chem. Sci., 2022, 13, 13303 DOI: 10.1039/D2SC05031E

This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. You can use material from this article in other publications without requesting further permissions from the RSC, provided that the correct acknowledgement is given.

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