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Molecular mechanism of the chitinolytic peroxygenase reaction

Significance Biomass decomposition by microorganisms relies on a battery of enzymes, of which lytic polysaccharide monooxygenases (LPMOs) constitute a central player. LPMOs are notably able to disrupt the abundant recalcitrant carbohydrates cellulose and chitin and thus increase the overall rate of their degradation. It has recently been demonstrated that LPMOs prefer hydrogen peroxide over molecular oxygen to oxidize and cleave their substrates. Here, we explain through molecular simulations and experiments how a chitin-active LPMO activates hydrogen peroxide and catalyzes substrate hydrogen abstraction. Thereby, we reveal the molecular details of this unusual bioinorganic chemistry, which is of high relevance for the understanding of pathogenesis-defense mechanisms and biomass turnover. Lytic polysaccharide monooxygenases (LPMOs) are a recently discovered class of monocopper enzymes broadly distributed across the tree of life. Recent reports indicate that LPMOs can use H2O2 as an oxidant and thus carry out a novel type of peroxygenase reaction involving unprecedented copper chemistry. Here, we present a combined computational and experimental analysis of the H2O2-mediated reaction mechanism. In silico studies, based on a model of the enzyme in complex with a crystalline substrate, suggest that a network of hydrogen bonds, involving both the enzyme and the substrate, brings H2O2 into a strained reactive conformation and guides a derived hydroxyl radical toward formation of a copper–oxyl intermediate. The initial cleavage of H2O2 and subsequent hydrogen atom abstraction from chitin by the copper–oxyl intermediate are the main energy barriers. Stopped-flow fluorimetry experiments demonstrated that the priming reduction of LPMO–Cu(II) to LPMO–Cu(I) is a fast process compared to the reoxidation reactions. Using conditions resulting in single oxidative events, we found that reoxidation of LPMO–Cu(I) is 2,000-fold faster with H2O2 than with O2, the latter being several orders of magnitude slower than rates reported for other monooxygenases. The presence of substrate accelerated reoxidation by H2O2, whereas reoxidation by O2 became slower, supporting the peroxygenase paradigm. These insights into the peroxygenase nature of LPMOs will aid in the development and application of enzymatic and synthetic copper catalysts and contribute to a further understanding of the roles of LPMOs in nature, varying from biomass conversion to chitinolytic pathogenesis-defense mechanisms.