Mapping the Initial Stages of a Protective Pathway that Enhances Catalytic Turnover by a Lytic Polysaccharide Monooxygenase

Abstract

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 stoppedflow spectroscopy, targeted mutagenesis, TD-DFT calculations, highenergy 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 Cu II −(histidyl radical), then reacts with a nearby tyrosine residue in an intersystem-crossing reaction to give a ferromagnetically coupled (S = 1) Cu II −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.

Understanding the initial events of the oxidative damage and protection mechanisms of the AA9 lytic polysaccharide monooxygenase family

“…Reactions II and III were recently suggested as a protective hole-hopping mechanism. 42 From Fig. 9, we can see that Fig.…”

“…Spurred by the recent proposal of a histidyl radical 42 ( 3 ) as the first intermediate in a protective hole-hopping pathway, we investigated whether the histidyl radical ( 3 ) can abstract a hydrogen from the tyrosine, thereby restoring histidine while forming a tyrosyl radical ( 2 ). This reaction is labeled III in Fig.…”

“…This commensurates with experiments for both Ta AA9 and Ls AA9, were a number of studies detected 2 (ref. 36–38 and 42) for different LPMOs, including Ta AA9 (ref. 36) and Ls AA9.…”

“…In a previous paper 40 we compared calculated UV-vis spectra with the characteristic observed bands around 400–420 nm for 2 , 36–38,42 but our calculations in ref. 40 were exclusively done on Ls AA9.…”

“…The last open question arises since previous QM/MM calculations on the protective or oxidative damage mechanisms focused exclusively on a particular member of the AA9 family, namely LsAA9, 35,40 whereas experimental studies focused on different LPMOs. 36,39,42 In this paper we will address (i) and (ii) by employing QM/MM calculations for reactions I-III in Fig. 1.…”