Histidine oxidation in lytic polysaccharide monooxygenase

Abstract

The lytic polysaccharide monooxygenases (LPMOs) comprise a super-family of copper enzymes that boost the depolymerisation of polysaccharides by oxidatively disrupting the glycosidic bonds connecting the sugar units. Industrial use of LPMOs for cellulose depolymerisation has already begun but is still far from reaching its full potential. One issue is that the LPMOs self-oxidise and thereby deactivate. The mechanism of this self-oxidation is unknown, but histidine residues coordinating to the copper atom are the most susceptible. An unusual methyl modification of the NE2 atom in one of the coordinating histidine residues has been proposed to have a protective role. Furthermore, substrate binding is also known to reduce oxidative damage. We here for the first time investigate the mechanism of histidine oxidation with combined quantum and molecular mechanical (QM/MM) calculations, with outset in intermediates previously shown to form from a reaction with peroxide and a reduced LPMO. We show that an intermediate with a [Cu–O]+ moiety is sufficiently potent to oxidise the nearest C–H bond on both histidine residues, but methylation of the NE2 atom of His-1 increases the reaction barrier of this reaction. The substrate further increases the activation barrier. We also investigate a [Cu–OH]2+ intermediate with a deprotonated tyrosine radical. This intermediate was previously proposed to have a protective role, and we also find it to have higher barriers than the corresponding a [Cu–O]+ intermediate. Graphical abstract

Kβ X-ray Emission Spectroscopy of Cu(I)-Lytic Polysaccharide Monooxygenase: Direct Observation of the Frontier Molecular Orbital for H2O2 Activation

“…21,22 Several possible roles of the His-brace have been proposed but its function is still unknown. 20,23,24 Some LPMO families exhibit methylation of the His-brace residue at Nε2 (including the AA9 family containing Hypocrea jecorina LPMO9A (HjLPMO9A) studied here). However, this methylation does not appear to significantly impact reactivity relative to LPMOs without this methylation.…”

“…Across all LPMO families, the Cu­(I) is bound by two trans histidine residues, one being the N-terminal His that binds bidentate through both its amine and imidazole groups forming a “His-brace” motif (Figure , inset) . This leads to a square planar geometry with an open coordination position trans to the amine of the His-brace. ,, This His-brace motif is also found in copper binding proteins and particulate methane monooxygenase (pMMO), although recent literature suggests the His-brace is not the catalytically active site in pMMO. , Several possible roles of the His-brace have been proposed but its function is still unknown. ,, Some LPMO families exhibit methylation of the His-brace residue at Nε2 (including the AA9 family containing Hypocrea jecorina LPMO9A ( Hj LPMO9A) studied here). However, this methylation does not appear to significantly impact reactivity relative to LPMOs without this methylation. , Although Cu­(I)-LPMO can oxidize substrate in the presence of O 2 or H 2 O 2 , the reaction of Cu­(I)-LPMO with H 2 O 2 in the absence of substrate has been found to be 3 orders of magnitude faster , than with O 2 .…”

“…This leads to a square planar geometry with an open coordination position trans to the amine of the His-brace. ,, This His-brace motif is also found in copper binding proteins and particulate methane monooxygenase (pMMO), although recent literature suggests the His-brace is not the catalytically active site in pMMO. , Several possible roles of the His-brace have been proposed but its function is still unknown. ,, Some LPMO families exhibit methylation of the His-brace residue at Nε2 (including the AA9 family containing Hypocrea jecorina LPMO9A ( Hj LPMO9A) studied here). However, this methylation does not appear to significantly impact reactivity relative to LPMOs without this methylation. , Although Cu­(I)-LPMO can oxidize substrate in the presence of O 2 or H 2 O 2 , the reaction of Cu­(I)-LPMO with H 2 O 2 in the absence of substrate has been found to be 3 orders of magnitude faster , than with O 2 . Certain species of LPMO have been shown to have little activity in the absence of H 2 O 2 , , but enzyme inactivation can also occur from reaction with excess H 2 O 2 . , The H 2 O 2 reaction with Cu­(I)-LPMO generates a Cu­(II)–OH (from EPR) and an • OH radical (from radical trapping) indicating homolytic cleavage of the H 2 O 2 bond.…”

“…However, this methylation does not appear to significantly impact reactivity relative to LPMOs without this methylation. 5,23 Although Cu(I)-LPMO can oxidize substrate in the presence of O 2 or H 2 O 2 , 25 the reaction of Cu(I)-LPMO with H 2 O 2 in the absence of substrate has been found to be 3 orders of magnitude faster 7,26 than with O 2 . Certain species of LPMO have been shown to have little activity in the absence of H 2 O 2 , 27,28 but enzyme inactivation can also occur from reaction with excess H 2 O 2 .…”

A Conserved Second Sphere Residue Tunes Copper Site Reactivity in Lytic Polysaccharide Monooxygenases

“…Since the discovery of the LPMO reaction in 2010, headway has been made in elucidating the catalytic mechanism of these enzymes, and based on computational studies, it is now generally believed that the reactive copper species involved in hydrogen atom abstraction is a Cu­(II)-oxyl species. − Hydrogen atom abstraction by the Cu­(II)-oxyl is followed by a rebound of the Cu-bound hydroxyl, leading to substrate hydroxylation and destabilization of the glycosidic bond. ,, Reduction of LPMOs is essential for the enzymes to become catalytically competent and can be achieved by a variety of small reductants or electron-delivering enzymes. , While LPMOs were originally thought to proceed via a monooxygenase mechanism (R–H + O 2 + 2e – + 2H + → R–OH + H 2 O), evidence strongly suggests that under most, if not all, conditions, LPMOs catalyze a peroxygenase reaction (R–H + H 2 O 2 → R – OH + H 2 O). ,, The peroxygenase reaction is orders of magnitude faster than the apparent monooxygenase reaction. − , It is believed that LPMOs source H 2 O 2 through an intrinsic oxidase activity, through autoxidation of low molecular weight reductants typically used in LPMO reactions, through exogenous oxidases, or through abiotic H 2 O 2 generating sources such as irradiated lignin found in the same ecological niche. Predominant suggestions for the mechanism of the peroxygenase reaction entail initial homolytic cleavage of H 2 O 2 by the reduced LPMO yielding a Cu­(II)-hydroxide and a hydroxyl radical, where it is believed the latter can damage the LPMO if the LPMO is not bound to substrate to engage in productive chemistry. ,− …”