Spectroscopic and computational insight into the activation of O ₂ by the mononuclear Cu center in polysaccharide monooxygenases

Abstract

Strategies for O 2 activation by copper enzymes were recently expanded to include mononuclear Cu sites, with the discovery of the copper-dependent polysaccharide monooxygenases, also classified as auxiliary-activity enzymes 9-11 (AA9-11). These enzymes are finding considerable use in industrial biofuel production. Crystal structures of polysaccharide monooxygenases have emerged, but experimental studies are yet to determine the solution structure of the Cu site and how this relates to reactivity. From X-ray absorption near edge structure and extended X-ray absorption fine structure spectroscopies, we observed a change from four-coordinate Cu(II) to three-coordinate Cu(I) of the active site in solution, where three protein-derived nitrogen ligands coordinate the Cu in both redox states, and a labile hydroxide ligand is lost upon reduction. The spectroscopic data allowed for density functional theory calculations of an enzyme active site model, where the optimized Cu(I) and (II) structures were consistent with the experimental data. The O 2 reactivity of the Cu(I) site was probed by EPR and stopped-flow absorption spectroscopies, and a rapid one-electron reduction of O 2 and regeneration of the resting Cu(II) enzyme were observed. This reactivity was evaluated computationally, and by calibration to Cu-superoxide model complexes, formation of an end-on Cu-AA9-superoxide species was found to be thermodynamically favored. We discuss how this thermodynamically difficult one-electron reduction of O 2 is enabled by the unique protein structure where two nitrogen ligands from His1 dictate formation of a T-shaped Cu(I) site, which provides an open coordination position for strong O 2 binding with very little reorganization energy.X-ray absorption spectroscopy | DFT | dioxygen activation | biofuels

Oxygen Activation at the Active Site of a Fungal Lytic Polysaccharide Monooxygenase

“…[4] Dioxo coordination in the available equatorial coordination site is also consistent with a classical associative displacement mechanism for hydroperoxo release by an axially coordinated water molecule in the absence of polysaccharide substrate. [9] The equatorial dioxo coordination observed is different from that modeled in the previous NcPMO-2 crystal structure (see Fig.…”

“…[4,14] We performed DFT geometry optimizations of three active site models (ASMs) derived from our high-resolution resting state X-ray structure and calculated the free energy of molecular O 2 "pre-binding" when His157 exhibits different side chain conformations and protonation states. (All atoms of the ASMs are shown in Fig.…”

Structural and Functional Characterization of a Lytic Polysaccharide Monooxygenase with Broad Substrate Specificity

“…A similar difference has been observed when comparing a strictly C1-oxidizing LPMO10 with an LPMO10 that can oxidize both C1 and C4 (17). It has been shown that end-on binding of dioxygen is energetically favorable (12,49); however, although Kim et al (49) model O 2 binding at the exposed axial position, Kjaergaard et al (12) propose a model where the equatorial OH ligand is replaced by O 2 . This discrepancy is probably a result of different initial structural models and basis sets used in the density functional theory (DFT) calculations.…”

“…Although binding of multiple bivalent metal ions has been observed in the crystal structures for several LPMOs (5,17), available experimental and theoretical data consistently indicate that the active site copper centers are mononuclear (11,12,17,49). The additional metal ions observed in the zinc structure of NcLPMO9C are coordinated by amino acids belonging to a sequence insertion unique for strictly C4-oxidizing LPMO9s, but Fig.…”

“…Electrons may be provided by a reducing agent such as ascorbic acid, gallate, or reduced glutathione (5,6) or by a co-secreted enzyme such as cellobiose dehydrogenase (10). The catalytic copper-binding site of LPMOs has unique features that are shared with only one other type of enzyme, methane monooxygenase (5,11,12).…”