Kinetic analysis of amino acid radicals formed in H ₂ O ₂ -driven Cu ^I LPMO reoxidation implicates dominant homolytic reactivity

Abstract

Lytic polysaccharide monooxygenases (LPMOs) have been proposed to react with bothO2andH2O2as cosubstrates. In this study, theH2O2reaction with reducedHypocrea jecorinaLPMO9A (CuI-HjLPMO9A) is demonstrated to be 1,000-fold faster than theO2reaction while producing the same oxidized oligosaccharide products. Analysis of the reactivity in the absence of polysaccharide substrate by stopped-flow absorption and rapid freeze–quench (RFQ) electron paramagnetic resonance (EPR) and magnetic circular dichroism (MCD) yields two intermediates corresponding to neutral tyrosyl and tryptophanyl radicals that are formed along minor reaction pathways. The dominant reaction pathway is characterized by RFQ EPR and kinetic modeling to directly produce CuII-HjLPMO9A and indicates homolytic O–O cleavage. Both optical intermediates exhibit magnetic exchange coupling with the CuIIsites reflecting facile electron transfer (ET) pathways, which may be protective against uncoupled turnover or provide an ET pathway to the active site with substrate bound. The reactivities of nonnative organic peroxide cosubstrates effectively exclude the possibility of a ping-pong mechanism.

Genomic and transcriptomic analysis of the thermophilic lignocellulose-degrading fungus Thielavia terrestris LPH172

“…Its upregulation on Avicel could, instead, be explained by coregulation of cellulolytic and lignocellulolytic enzymes. Either way, AA4s can produce H 2 O 2 , which is also a co-substrate for LPMOs (75)(76)(77). In the case of high LPMO expression and catalysis, it is conceivable that the fungus tries to produce enough co-substrate for all its LPMOs.…”

A thermostable bacterial lytic polysaccharide monooxygenase with high operational stability in a wide temperature range

“…LPMO catalysis has generally been thought to be strictly dependent on molecular oxygen and a reductant that delivers two electrons and two protons for each catalytic cycle [9,23,24]. However, recent studies on LPMOs belonging to families AA9 and AA10, have shown that H 2 O 2 can drive LPMO reactions, and that these reactions are orders of magnitude faster than O 2 -driven reactions [20,[25][26][27][28][29]. The peroxygenase driven reaction only requires sub-stoichiometric amounts of reductant for an initial, "priming", reduction of the LPMO, after which the enzyme can catalyze multiple reactions.…”

Kinetic insights into the peroxygenase activity of cellulose-active lytic polysaccharide monooxygenases (LPMOs)

“…The scarcity of kinetic data likely reflects the numerous experimental challenges associated with quantitative characterization of LPMO functionality 12 . The complexity is exemplified by the recent discovery that LPMOs may use H 2 O 2 , rather than, or even instead of, O 2 , as co-substrate [13][14][15][16][17][18][19][20] . Regardless of the mechanism, LPMOs need an external electron donor for catalysis.…”