The Copper Active Site of CBM33 Polysaccharide Oxygenases

Abstract

The capacity of metal-dependent fungal and bacterial polysaccharide oxygenases, termed GH61 and CBM33, respectively, to potentiate the enzymatic degradation of cellulose opens new possibilities for the conversion of recalcitrant biomass to biofuels. GH61s have already been shown to be unique metalloenzymes containing an active site with a mononuclear copper ion coordinated by two histidines, one of which is an unusual τ-N-methylated N-terminal histidine. We now report the structural and spectroscopic characterization of the corresponding copper CBM33 enzymes. CBM33 binds copper with high affinity at a mononuclear site, significantly stabilizing the enzyme. X-band EPR spectroscopy of Cu(II)-CBM33 shows a mononuclear type 2 copper site with the copper ion in a distorted axial coordination sphere, into which azide will coordinate as evidenced by the concomitant formation of a new absorption band in the UV/vis spectrum at 390 nm. The enzyme’s three-dimensional structure contains copper, which has been photoreduced to Cu(I) by the incident X-rays, confirmed by X-ray absorption/fluorescence studies of both aqueous solution and intact crystals of Cu-CBM33. The single copper(I) ion is ligated in a T-shaped configuration by three nitrogen atoms from two histidine side chains and the amino terminus, similar to the endogenous copper coordination geometry found in fungal GH61.

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

“…In the first fast scan of Cu(II)-AA9 (blue trace) (radiation dose: ∼1.7 × 10 12 photons/mm 2 ), very little intensity at 8984 eV is seen, verifying the presence of the oxidized state of the Cu. However, the oxidized enzyme undergoes photoreduction in the beam as observed in the average of longer scans from four fresh spots (black dashed trace) (radiation dose: ∼6.6 × 10 12 photons/mm 2 per scan), similar to observations for AA10 PMOs (17). In contrast, the spectrum of the fully reduced state (red trace, Fig.…”

“…While no redox potential has been reported for AA9, the redox potential of AA10 enzymes has been reported at ∼275 mV vs. the normal hydrogen electrode (NHE) (15,17). Using this potential, and the potential of the one-electron reduction of O 2 to superoxide (−165 mV vs. NHE) (27), the outer-sphere electron transfer rate from Cu(I) to O 2 is estimated to be ∼4.5 × 10 −4 s −1 (see SI Appendix) which is ∼10 3 slower than the experimental rate of Cu(I) reoxidation from the EPR and stopped-flow data.…”

“…Although little is known about the reaction mechanism of these enzymes, it is believed that this does not simply involve Fenton-type chemistry producing reactive oxygen species but rather a more controlled reaction involving a Cu-oxygen intermediate (14)(15)(16). Cu-loaded crystal structures have been solved for all three subclasses of AA9-11 enzymes (7,8,(17)(18)(19), highlighting interesting similarities and differences. Common for the three classes is a flat protein surface that harbors the Cu active site, as illustrated for an AA9 enzyme in Fig.…”

“…Although the crystal structures provide valuable information, they do not determine the detailed environment of the Cu ion in solution, which is critical in elucidating the mechanistic properties of the PMOs. This is partly due to the fact that oxidized Cu readily undergoes photoreduction upon exposure to X-rays, often accompanied by changes in the coordination environment of the metal (17,20).…”

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

“…However, it has been shown recently that oxidative degradation catalyzed by lytic polysaccharide monooxygenases (LPMOs) 5 (1) plays a significant role (2)(3)(4)(5)(6). LPMOs are copperdependent biocatalysts that use molecular oxygen and an electron donor to break glycosidic bonds (5)(6)(7)(8)(9). 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).…”