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

Abstract

Lytic polysaccharide monooxygenases (LPMOs) are powerful monocopper enzymes that can activate strong C–H bonds through a mechanism that remains largely unknown. Herein, we investigated the role of a conserved glutamine/glutamate in the second coordination sphere. Mutation of the Gln in NcAA9C to Glu, Asp, or Asn showed that the nature and distance of the headgroup to the copper fine-tune LPMO functionality and copper reactivity. The presence of Glu or Asp close to the copper lowered the reduction potential and decreased the ratio between the reduction and reoxidation rates by up to 500-fold. All mutants showed increased enzyme inactivation, likely due to changes in the confinement of radical intermediates, and displayed changes in a protective hole-hopping pathway. Electron paramagnetic resonance (EPR) and X-ray absorption spectroscopic (XAS) studies gave virtually identical results for all NcAA9C variants, showing that the mutations do not directly perturb the Cu(II) ligand field. DFT calculations indicated that the higher experimental reoxidation rate observed for the Glu mutant could be reconciled if this residue is protonated. Further, for the glutamic acid form, we identified a Cu(III)-hydroxide species formed in a single step on the H2O2 splitting path. This is in contrast to the Cu(II)-hydroxide and hydroxyl intermediates, which are predicted for the WT and the unprotonated glutamate variant. These results show that this second sphere residue is a crucial determinant of the catalytic functioning of the copper-binding histidine brace and provide insights that may help in understanding LPMOs and LPMO-inspired synthetic catalysts.

Electrochemical Monitoring of Heterogeneous Peroxygenase Reactions Unravels LPMO Kinetics

“…In contrast, the second-order rate constant for the reoxidation of NcAA9C−Cu(I) shows no pHdependency (Figure S20). Notably, the obtained second-order rate constants for reduction and reoxidation at pH 7.0 are in line with data reported by Hall et al 43 The fast kinetic data are in accord with the pH-dependency of half-saturating ascorbate concentrations derived from steady-state analysis (Figure 4C and Table S10). After finding that the ascorbic acid/ascorbate concentration necessary to reach half-maximal activity is ∼50-fold higher at pH 4.0 than at pH 7.0 (890 and 17 μM, respectively), a reassessment of the pH profile using increased ascorbate concentrations showed essentially no pH-dependency of LPMO activity between pH 4.0 and 6.0 (TN = 30−35 s −1 ), while activity still increases with pH from 6.0 to 8.0 (TN = 35−55 s −1 ) (Figure 4A).…”

“…In contrast, the second-order rate constant for the reoxidation of Nc AA9C–Cu­(I) shows no pH-dependency (Figure S20). Notably, the obtained second-order rate constants for reduction and reoxidation at pH 7.0 are in line with data reported by Hall et al…”

Impact of the Copper Second Coordination Sphere on Catalytic Performance and Substrate Specificity of a Bacterial Lytic Polysaccharide Monooxygenase

“…The increased oxidase activity that results from the Q219E mutation at position 2 relies on the presence of a tyrosine at position 3, and future studies should aim to uncover the relationship between these two residues. Interestingly, the interplay between residues at positions 2 and 3 could couple the variation at position 2 to possible protective hole hopping mechanisms mediated by a tyrosine at position 3, ,, as recently shown for a cellulose-active AA9 LPMO carrying the HQY motif . On a related note, the presence of a tyrosine at position 3 and glutamate at position 2 does not exist naturally in AA10 LPMOs but is the most prevalent combination in (chitin-active) LPMOs in the AA11 family.…”

“…Another conserved second sphere residue is a glutamine or glutamate residue, which typically coexists with an axial tyrosine or phenylalanine, respectively, in AA9 and AA10 LPMOs. This glutamine/glutamate residue has been shown to play a crucial role in the peroxygenase reaction through constraining and orienting H 2 O 2 and subsequent reactive intermediates. ,, Mutagenesis studies have confirmed this, along with an additional role in controlling copper reactivity. , Alongside the glutamine in AA9s, a conserved histidine residue has also been predicted to play a role in positioning oxygen species and has a debated role as a proton donor. ,− …”

Mimicking the Reactivity of LPMOs with a Mononuclear Cu Complex

“…[7] DeBeer, Eijsink, Sørlie and coworkers have also recently reported that the protonation state of a conserved glutamine/glutamate residue on the secondary coordination sphere of the Cu active center might also play a critical role on controlling the oxidase, peroxidase and peroxygenase reactivity. [8] In the peroxygenase and peroxidase reactions, the proposed catalytic cycles are initiated by coordination of the H 2 O 2 to the Cu I ion, which is proposed to trigger an homolytic OÀ O bond cleavage to generate a Cu II OH core and hydroxyl radical (species G and H in Figure 1B). [9] In the peroxygenase cycle, the reaction between the CuOH core and the hydroxyl radical is proposed to generate the hydroxylating Cu-oxyl intermediate (species G to species E in Figure 1B), a reaction pathway supported by computations.…”