Abstract
Hydrogen peroxide is a cosubstrate for the oxidative cleavage of saccharidic substrates by copper-containing lytic polysaccharide monooxygenases (LPMOs). The rate of reaction of LPMOs with hydrogen peroxide is high, but it is accompanied by rapid inactivation of the enzymes, presumably through protein oxidation. Herein, we use UV–vis, CD, XAS, EPR, VT/VH-MCD, and resonance Raman spectroscopies, augmented with mass spectrometry and DFT calculations, to show that the product of reaction of an AA9 LPMO with H2O2 at higher pHs is a singlet Cu(II)–tyrosyl radical species, which is inactive for the oxidation of saccharidic substrates. The Cu(II)–tyrosyl radical center entails the formation of significant Cu(II)–(●OTyr) overlap, which in turn requires that the plane of the d(x2–y2) SOMO of the Cu(II) is orientated toward the tyrosyl radical. We propose from the Marcus cross-relation that the active site tyrosine is part of a “hole-hopping” charge-transfer mechanism formed of a pathway of conserved tyrosine and tryptophan residues, which can protect the protein active site from inactivation during uncoupled turnover.
Highlights
Lytic polysaccharide monooxygenases (LPMOs, known as PMOs) are copper-containing enzymes that catalyze the oxidative cleavage of polysaccharides by dioxygen or hydrogen peroxide.[1]
None of the preferred pathways from these calculations invokes a role for the tyrosine residue within the catalytic mechanism.[24]. It is in this context that we report a multispectroscopic (EPR, VT/VH-magnetic circular dichroism (MCD), CD, UV−vis, XAS, resonance Raman), mass spectrometry, and density functional theory (DFT) study into a purple-colored species that arises during the uncoupled turnover24c,25 of an
In addition to O2 acting as a cosubstrate, it has recently been reported that hydrogen peroxide acts as a cosubstrate for lytic polysaccharide monooxygenases (LPMOs), replacing the combination of O2 and reducing agent, albeit in a reaction which is accompanied by significant protein degradation.1c,28 Despite the fact that the reaction with peroxide is deleterious to the enzyme, the addition of peroxide to LPMOs provides for a potential laboratory “shunt” that avoids the complicating use of reducing agents within spectroscopic and activity studies.24c taking advantage of the peroxide shunt reaction with LPMOs, we added various concentrations of hydrogen peroxide to ∼1 mM solutions of an AA9 LPMO from Lentinus similis (LsAA9) which had previously been spectroscopically and structurally characterized.[2,29]
Summary
Lytic polysaccharide monooxygenases (LPMOs, known as PMOs) are copper-containing enzymes that catalyze the oxidative cleavage of polysaccharides by dioxygen or hydrogen peroxide.[1] The active site of LPMOs contains a single copper ion coordinated by an N-terminal histidine through the NH2 of the amino terminus and the π-N of its imidazole side chain.[2] A T-shaped coordination geometry at the Cu is completed by the τ-N atom of a further histidine side chain. This structural unit is known as the histidine brace[3] (Scheme 1).
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