The Role of the Secondary Coordination Sphere in a Fungal Polysaccharide Monooxygenase

  • Sphere Stabilization: H161 and Q167 form a hydrogen-bond network that stabilizes bound oxygen and promotes proton transfer.
  • Motif Variability: Two motifs (REF and HQY/HQF) dominate second-sphere composition across cellulose-active AA10s, reflecting evolutionary tuning.
  • Protonation Tuning: Mutation of conserved glutamine/glutamate shifts reduction potentials by up to 500 mV, altering reduction/reoxidation kinetics.
  • Substrate Recognition: Second-sphere histidine in LPMO9G facilitates cellulose binding and glycosidic bond destabilization at ~3 Å distances.
  • Peroxygenase Enhancement: Q219E mutation boosts chitin oxidation by increasing in situ H₂O₂ generation under reductant-driven conditions.
  • Solvent Rescue: Even disruptive alanine substitutions retain partial activity via solvent penetrating the cavity, highlighting sphere adaptability.
  1. The Role of the Secondary Coordination Sphere in a Fungal Polysaccharide Monooxygenase: Marletta, M.A., et al., ACS Chem. Biol., 2017
  2. Impact of the Copper Second Coordination Sphere on Catalytic Fine-Tuning of LPMOs: Vaaje-Kolstad, G., et al., ACS Omega, 2024
  3. A Conserved Second Sphere Residue Tunes Copper Site Reactivity in LPMOs: Søgaard, D., et al., J. Am. Chem. Soc., 2023
  4. Assessing the Role of Redox Partners in TthLPMO9G and Its Mutants: Turner, C., et al., Front. Microbiol., 2024

Unveiling Hidden Helpers: How the Secondary Coordination Sphere Powers Fungal LPMOs

Critical hydrogen-bonding networks in fungal polysaccharide monooxygenases (PMOs) that govern oxygen activation and proton transfer—key mechanistic insights for optimizing enzymatic cellulose degradation, with transformative potential for biofuel production and sustainable biomass utilization.

From the moment copper binds within a fungal polysaccharide monooxygenase’s active site, a bustling network of neighboring amino acids leap into action—fine-tuning oxygen activation, stabilizing reactive intermediates, and orchestrating efficient degradation of cellulose. Recent studies spanning 2024–2025 have cast new light on this once–overlooked ensemble of residues, revealing how second-sphere players such as histidines, glutamines, and tyrosines amplify catalytic performance and broaden substrate selectivity. While the primary histidine brace secures the metal, it is this surrounding hydrogen-bonding web that truly unlocks the enzyme’s oxidative prowess, transforming what was once viewed as structural padding into a dynamic, integral partner in fungal biomass conversion.

Within the fungal AA9 family, mutational analyses of Myceliophthora thermophila PMO3* variants demonstrate that disrupting the H161–Q167 network slashes oxygen binding and proton-transfer efficiency—yet retains partial activity through solvent rescue, underscoring the sphere’s role in stabilizing a fleeting copper-oxyl intermediate. Complementary 2024 work on bacterial AA10 LPMOs reveals that swapping the His–Gln–Tyr motif for Arg–Glu–Phe accelerates oxidative inactivation under turnover, highlighting complex interplay between second-sphere composition and enzyme longevity. Moreover, advanced spectroscopy and DFT calculations on Neurospora crassa AA9C variants confirm that protonation states of a conserved glutamine/glutamate tune reduction potentials by hundreds of millivolts, modulating the ratio of reduction to reoxidation rates and controlling radical confinement pathways.

Beyond individual residues, bioinformatic surveys of over 460 predicted cellulose-active AA10s uncover two dominant second-sphere motifs—REF and HQY/HQF—equally represented across diverse bacterial lineages, suggesting evolutionary pressure to balance substrate affinity and oxidative stability. Detailed structural mapping in Thermothelomyces thermophilus LPMO9G further pinpoints a second-sphere histidine (H140) lying within 3.3 Å of the histidine brace, directly influencing cellulose recognition and glycosidic-bond destabilization. As peroxygenase functionality emerges, new mutagenesis of axial glutamine residues demonstrates enhanced chitin activity when fueled by exogenous H₂O₂, opening doors to tailored biocatalysts for polysaccharide valorization.

Collectively, these advances reveal the secondary coordination sphere not as passive support but as a dynamic regulator—safeguarding reactive species, directing proton flows, and sculpting substrate channels. By harnessing this hidden network, next-generation LPMO engineering can achieve unprecedented efficiency in biomass conversion, sustainable biofuel production, and precision biotransformations.

Concept Description Key Reference
Hydrogen-Bond Network H161 and Q167 stabilize oxygen binding, aiding proton transfer. Marletta, et al., ACS Chem. Biol., 2017
Motif Diversity REF and HQY/HQF motifs prevalent across AA10 LPMOs modulate reactivity. Vaaje-Kolstad, et al., ACS Omega, 2024
Redox Tuning Glutamine/glutamate protonation alters reduction potentials by hundreds of mV. Søgaard, et al., J. Am. Chem. Soc., 2023
Substrate Binding Second-sphere histidine at ~3.3 Å from the brace directs polysaccharide recognition. González-García, et al., Biochem. Soc. Trans., 2021
Peroxygenase Activity Q219E mutation elevates H₂O₂-driven chitin oxidation in AA10s. Vaaje-Kolstad, et al., ACS Omega, 2024
Solvent Rescue Cavity created by alanine substitutions allows solvent H-bonding to sustain activity. Marletta, et al., ACS Chem. Biol., 2017