The histidine-brace active site of a copper monooxygenase is redox active and forms part of an in-built enzyme repair mechanism

Abstract Oxygenase enzymes generate reactive intermediates at their active sites to effect controlled functionalizations 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 turnover in the absence of substrate. Herein we use a combination of stopped-flow spectroscopy, targeted mutagenesis, DFT calculations, high-energy resolution fluorescence detection X-ray absorption spectroscopy (HERFD-XAS), and electron paramagnetic resonance spectroscopy (EPR) to capture two transient intermediates that together form a protective pathway built into the active sites of copper-dependent lytic polysaccharide monooxygenases (LPMOs). First, a spin singlet (S = 0) CuII-(histidyl radical) is generated at the histidine brace active site following treatment of the LPMO with either hydrogen peroxide or peroxyacids in the absence of substrate. This intermediate 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 and the histidine brace active site to its resting state to facilitate resumption of the catalytic cycle through reduction. This process gives the enzyme the capacity to repair any damage to the active site histidine residues ‘on the fly’, highlighting how enzymes protect themselves from deleterious side reactions during uncoupled turnover.