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Oxygenase and peroxygenase enzymes generate intermediates at their active sites which bring about the controlled functionalization of inert C–H bonds in substrates, such as in the enzymatic conversion of methane to methanol. To be viable catalysts, however, these enzymes must also prevent oxidative damage to essential active site residues, which can occur during both coupled and uncoupled turnover. Herein, we use a combination of stopped-flow spectroscopy, targeted mutagenesis, TD-DFT calculations, high-energy resolution fluorescence detection X-ray absorption spectroscopy, and electron paramagnetic resonance spectroscopy to study two transient intermediates that together form a protective pathway built into the active sites of copper-dependent lytic polysaccharide monooxygenases (LPMOs). First, a transient high-valent species is generated at the copper histidine brace active site following treatment of the LPMO with either hydrogen peroxide or peroxyacids in the absence of substrate. This intermediate, which we propose to be a CuII–(histidyl radical), then reacts with a nearby tyrosine residue in an intersystem-crossing reaction to give a ferromagnetically coupled (S= 1) CuII–tyrosyl radical pair, thereby restoring the histidine brace active site to its resting state and allowing it to re-enter the catalytic cycle through reduction. This process gives the enzyme the capacity to minimize damage to the active site histidine residues “on the fly” to increase the total turnover number prior to enzyme deactivation, highlighting how oxidative enzymes are evolved to protect themselves from deleterious side reactions during uncoupled turnover.

The flavin and heme cofactor containing enzyme cellobiose dehydrogenase (CDH) is ubiquitously distributed in wood-degrading fungi. Current research provides compelling evidence that CDH is an activator for cellulolytic monooxygenases, which enhance the accessibility of crystalline cellulose surfaces for hydrolases. Such oxidative cellulose degradation contributes to the overall cellulolytic capabilities of wood decaying fungi to a large extent, and holds great potential to improve the efficiency of commercial enzyme mixtures for biomass processing and biofuel production. This review summarizes current literature with regard to the distribution, structure and physiological role of CDH in the light of recent findings.

Uniting the unparalleled stereoselectivity of enzymes with the efficiency of chemical catalysis offers high product yields and atom economy while limiting downstream processing efforts. However, despite an impressive series of proof-of-concept studies, the field has not yet achieved broad applicability, especially in regard to industrial requirements. Although the focus has been mostly on proof-of-concept studies with well-established model enzymes such as lipases, transaminases, and alcohol dehydrogenases, we expect that the catalytic diversity will soon increase. However, this means that several challenges such as the stability and compatibility of the catalysts, their preference for different solvents, and their possible cross-reactivities in complex reactions need to be tackled. A powerful strategy to overcome these frequently observed ‘incompatibility problems’ of multistep cascade reactions is by physical separation of individual reaction steps through compartmentalization.