Active-site copper reduction promotes substrate binding of fungal lytic polysaccharide monooxygenase and reduces stability

Lytic polysaccharide monooxygenases (LPMOs) are a class of copper-containing enzymes that oxidatively degrade insoluble plant polysaccharides and soluble oligosaccharides. Upon reductive activation, they cleave the substrate and promote biomass degradation by hydrolytic enzymes. In this study, we employed LPMO9C from Neurospora crassa, which is active toward cellulose and soluble β-glucans, to study the enzyme-substrate interaction and thermal stability. Binding studies showed that the reduction of the mononuclear active-site copper by ascorbic acid increased the affinity and the maximum binding capacity of LPMO for cellulose. The reduced redox state of the active-site copper and not the subsequent formation of the activated oxygen species increased the affinity toward cellulose. The lower affinity of oxidized LPMO could support its desorption after catalysis and allow hydrolases to access the cleavage site. It also suggests that the copper reduction is not necessarily performed in the substrate-bound state of LPMO. Differential scanning fluorimetry showed a stabilizing effect of the substrates cellulose and xyloglucan on the apparent transition midpoint temperature of the reduced, catalytically active enzyme. Oxidative auto-inactivation and destabilization were observed in the absence of a suitable substrate. Our data reveal the determinants of LPMO stability under turnover and non-turnover conditions and indicate that the reduction of the active-site copper initiates substrate binding.


Figure S1
Nc sphere. and after addition of ascorbic acid to a final concentration of 5 mM (dashed lines). Spectra were recorded Figure S2. Emission spectra of ANS recorded at 63 carried out in 50 mM phosphate buffer, pH 6 200 fluorescence intensities decreased spectra excitation at 378 nm.

Figure S1
NcLPMO9C sphere. and after addition of ascorbic acid to a final concentration of 5 mM (dashed lines). Spectra were recorded Figure S2. Emission spectra of ANS recorded at 63 carried out in 50 mM phosphate buffer, pH 6 200 µ fluorescence intensities decreased spectra excitation at 378 nm.

Figure S1
LPMO9C sphere. B and after addition of ascorbic acid to a final concentration of 5 mM (dashed lines). Spectra were recorded Figure S2. Emission spectra of ANS recorded at 63 carried out in 50 mM phosphate buffer, pH 6 µM ANS, which is a 40 fluorescence intensities decreased spectra from spectra of excitation at 378 nm. Figure S1.
LPMO9C B, Excitation and emission and after addition of ascorbic acid to a final concentration of 5 mM (dashed lines). Spectra were recorded at 30 °C Figure S2. Emission spectra of ANS recorded at 63 carried out in 50 mM phosphate buffer, pH 6 M ANS, which is a 40 fluorescence intensities decreased from spectra of excitation at 378 nm.
. Molecular structure and fluorescence spectr LPMO9C (pdb: 4D7U) , Excitation and emission and after addition of ascorbic acid to a final concentration of 5 mM (dashed lines). Spectra were at 30 °C Figure S2. Emission spectra of ANS recorded at 63 carried out in 50 mM phosphate buffer, pH 6 M ANS, which is a 40 fluorescence intensities decreased from spectra of excitation at 378 nm.
Molecular structure and fluorescence spectr (pdb: 4D7U) , Excitation and emission and after addition of ascorbic acid to a final concentration of 5 mM (dashed lines). Spectra were at 30 °C Figure S2. Emission spectra of ANS recorded at 63 °C, where highest carried out in 50 mM phosphate buffer, pH 6 M ANS, which is a 40 fluorescence intensities decreased from spectra of excitation at 378 nm.
Molecular structure and fluorescence spectr (pdb: 4D7U) , Excitation and emission and after addition of ascorbic acid to a final concentration of 5 mM (dashed lines). Spectra were at 30 °C in 50 mM sodium Figure S2. Emission spectra of ANS°C , where highest carried out in 50 mM phosphate buffer, pH 6 M ANS, which is a 40 fluorescence intensities decreased from spectra of excitation at 378 nm.
Molecular structure and fluorescence spectr (pdb: 4D7U) , Excitation and emission and after addition of ascorbic acid to a final concentration of 5 mM (dashed lines). Spectra were in 50 mM sodium Figure S2. Emission spectra of ANS°C , where highest carried out in 50 mM phosphate buffer, pH 6 M ANS, which is a 40 fluorescence intensities decreased from spectra of excitation at 378 nm.
Molecular structure and fluorescence spectr (pdb: 4D7U) , Excitation and emission and after addition of ascorbic acid to a final concentration of 5 mM (dashed lines). Spectra were in 50 mM sodium Figure S2. Emission spectra of ANS°C , where highest carried out in 50 mM phosphate buffer, pH 6 M ANS, which is a 40 fluorescence intensities decreased from spectra of LPMO Molecular structure and fluorescence spectr (pdb: 4D7U) with indicated tryptophan residues. , Excitation and emission and after addition of ascorbic acid to a final concentration of 5 mM (dashed lines). Spectra were in 50 mM sodium Figure S2. Emission spectra of ANS°C , where highest carried out in 50 mM phosphate buffer, pH 6 M ANS, which is a 40 fluorescence intensities decreased LPMO Molecular structure and fluorescence spectr with indicated tryptophan residues. , Excitation and emission and after addition of ascorbic acid to a final concentration of 5 mM (dashed lines). Spectra were in 50 mM sodium Figure S2. Emission spectra of ANS°C , where highest carried out in 50 mM phosphate buffer, pH 6 M ANS, which is a 40-fold molar excess over fluorescence intensities decreased LPMO-Molecular structure and fluorescence spectr with indicated tryptophan residues. , Excitation and emission and after addition of ascorbic acid to a final concentration of 5 mM (dashed lines). Spectra were in 50 mM sodium Figure S2. Emission spectra of ANS°C , where highest fluorescence carried out in 50 mM phosphate buffer, pH 6 fold molar excess over fluorescence intensities decreased -ANS complexes Molecular structure and fluorescence spectr with indicated tryptophan residues. , Excitation and emission spectr and after addition of ascorbic acid to a final concentration of 5 mM (dashed lines). Spectra were in 50 mM sodium phosphate Figure S2. Emission spectra of ANSfluorescence carried out in 50 mM phosphate buffer, pH 6 fold molar excess over slig ANS complexes Molecular structure and fluorescence spectr with indicated tryptophan residues. spectr and after addition of ascorbic acid to a final concentration of 5 mM (dashed lines). Spectra were phosphate -LPMO complexes. fluorescence carried out in 50 mM phosphate buffer, pH 6 fold molar excess over slightly ANS complexes Molecular structure and fluorescence spectr with indicated tryptophan residues. spectra of and after addition of ascorbic acid to a final concentration of 5 mM (dashed lines). Spectra were phosphate LPMO complexes. fluorescence carried out in 50 mM phosphate buffer, pH 6 fold molar excess over htly.

ANS complexes
Molecular structure and fluorescence spectr with indicated tryptophan residues. of Nc and after addition of ascorbic acid to a final concentration of 5 mM (dashed lines). Spectra were phosphate buffer, pH 6 LPMO complexes. fluorescence carried out in 50 mM phosphate buffer, pH 6.0. Highest fold molar excess over . B, differential spectra obtained by subtraction of ANS ANS complexes Molecular structure and fluorescence spectr with indicated tryptophan residues. NcLPMO9C and after addition of ascorbic acid to a final concentration of 5 mM (dashed lines). Spectra were buffer, pH 6 LPMO complexes. fluorescence emission . Highest fold molar excess over , differential spectra obtained by subtraction of ANS ANS complexes.

Molecular structure and fluorescence spectr
with indicated tryptophan residues. LPMO9C and after addition of ascorbic acid to a final concentration of 5 mM (dashed lines). Spectra were buffer, pH 6 LPMO complexes. emission . Highest fold molar excess over , differential spectra obtained by subtraction of ANS . Maxim Molecular structure and fluorescence spectr with indicated tryptophan residues. LPMO9C and after addition of ascorbic acid to a final concentration of 5 mM (dashed lines). Spectra were buffer, pH 6 LPMO complexes. emission . Highest fold molar excess over Nc , differential spectra obtained by subtraction of ANS axim Molecular structure and fluorescence spectr with indicated tryptophan residues. LPMO9C and after addition of ascorbic acid to a final concentration of 5 mM (dashed lines). Spectra were buffer, pH 6.0 LPMO complexes. A emission intensity . Highest fluorescence NcLPMO9C , differential spectra obtained by subtraction of ANS aximal Molecular structure and fluorescence spectra of with indicated tryptophan residues. The active (10 and after addition of ascorbic acid to a final concentration of 5 mM (dashed lines). Spectra were .0, at A, fluorescence spectra intensity fluorescence LPMO9C , differential spectra obtained by subtraction of ANS al emission of Nc The active (10 µM) and after addition of ascorbic acid to a final concentration of 5 mM (dashed lines). Spectra were at a scan speed of , fluorescence spectra intensity fluorescence LPMO9C , differential spectra obtained by subtraction of ANS emission

NcLPMO9
The active M) and after addition of ascorbic acid to a final concentration of 5 mM (dashed lines). Spectra were a scan speed of , fluorescence spectra intensity was fluorescence LPMO9C. At higher ANS concentrations, , differential spectra obtained by subtraction of ANS emission LPMO9 The active M) in its oxidized state ( and after addition of ascorbic acid to a final concentration of 5 mM (dashed lines). Spectra were a scan speed of , fluorescence spectra was fluorescence intensities were observed using . At higher ANS concentrations, , differential spectra obtained by subtraction of ANS emission at 480 nm was observed LPMO9 The active-site copper is shown as blue in its oxidized state ( and after addition of ascorbic acid to a final concentration of 5 mM (dashed lines). Spectra were a scan speed of , fluorescence spectra was observed. Experiments were intensities were observed using . At higher ANS concentrations, , differential spectra obtained by subtraction of ANS at 480 nm was observed LPMO9C site copper is shown as blue in its oxidized state ( and after addition of ascorbic acid to a final concentration of 5 mM (dashed lines). Spectra were a scan speed of , fluorescence spectra observed. Experiments were intensities were observed using . At higher ANS concentrations, , differential spectra obtained by subtraction of ANS at 480 nm was observed C. A site copper is shown as blue in its oxidized state ( and after addition of ascorbic acid to a final concentration of 5 mM (dashed lines). Spectra were a scan speed of 10 , fluorescence spectra observed. Experiments were intensities were observed using . At higher ANS concentrations, , differential spectra obtained by subtraction of ANS at 480 nm was observed A, ribbon site copper is shown as blue in its oxidized state ( and after addition of ascorbic acid to a final concentration of 5 mM (dashed lines). Spectra were 10 nm s , fluorescence spectra of oxidized LPMO observed. Experiments were intensities were observed using . At higher ANS concentrations, , differential spectra obtained by subtraction of ANS at 480 nm was observed , ribbon site copper is shown as blue in its oxidized state ( and after addition of ascorbic acid to a final concentration of 5 mM (dashed lines). Spectra were nm s of oxidized LPMO observed. Experiments were intensities were observed using . At higher ANS concentrations, , differential spectra obtained by subtraction of ANS at 480 nm was observed , ribbon site copper is shown as blue in its oxidized state ( and after addition of ascorbic acid to a final concentration of 5 mM (dashed lines). Spectra were nm s -1 .
of oxidized LPMO observed. Experiments were intensities were observed using . At higher ANS concentrations, , differential spectra obtained by subtraction of ANS at 480 nm was observed , ribbon drawing site copper is shown as blue in its oxidized state (solid and after addition of ascorbic acid to a final concentration of 5 mM (dashed lines). Spectra were .
of oxidized LPMO observed. Experiments were intensities were observed using . At higher ANS concentrations, , differential spectra obtained by subtraction of ANS at 480 nm was observed drawing site copper is shown as blue solid and after addition of ascorbic acid to a final concentration of 5 mM (dashed lines). Spectra were of oxidized LPMO observed. Experiments were intensities were observed using . At higher ANS concentrations, , differential spectra obtained by subtraction of ANS at 480 nm was observed drawing site copper is shown as blue lines) and after addition of ascorbic acid to a final concentration of 5 mM (dashed lines). Spectra were of oxidized LPMO observed. Experiments were intensities were observed using . At higher ANS concentrations, , differential spectra obtained by subtraction of ANS at 480 nm was observed upon of site copper is shown as blue lines) and after addition of ascorbic acid to a final concentration of 5 mM (dashed lines). Spectra were of oxidized LPMO observed. Experiments were intensities were observed using . At higher ANS concentrations, , differential spectra obtained by subtraction of ANS upon of site copper is shown as blue lines) observed. Experiments were intensities were observed using . At higher ANS concentrations, , differential spectra obtained by subtraction of ANS upon Figure S3. Unfolding of NcLPMO9C (5 µM) in presence of ANS (200 µM) and ascorbic acid (ASC, 5 mM). A, raw traces showing fluorescence emission intensities for LPMO/ANS complexes in presence or absence of ascorbic acid. For blank-corrected traces, fluorescence of ANS/ascorbic acid or ANS was subtracted. B, first derivative of unfolding traces shown in A. Data from unfolding experiments are expressed as mean values (± SD) from three independent repeats. C, remaining ascorbic acid concentrations measured after completion of unfolding assays (30 °C -75 °C at a rate of 1 °C min -1 ). Ascorbic acid was measured by adding 20 µL of the sample solution to a solution of 300 µM 2,6-dichloroindophenol. The initial concentration of ascorbic acid was 5 mM (dashed line). Absorbance of 2,6-dichloroindophenol was recorded at 520 nm (ε 520 = 6.9 mM cm -1 ). Data are expressed as mean values (± SD) from three independent repeats.  Figure S5. Effect of ascorbic acid (ASC, 5 mM), superoxide dismutase (SOD, 0.15 µM) and catalase (CAT, 720 U mL -1 ) on the unfolding of LPMO. Figure S6. Photometric determination of NcLPMO9C in presence of ascorbic acid (ASC). A, UVvisible spectra of NcLPMO9C (5 µM) in 50 mM sodium phosphate buffer, pH 6.0 (blue line) and upon addition of 1 mM ascorbic acid (black line). Spectra were recorded at room temperature (~23 °C) using a diode array spectrophotometer. Absorbance of ascorbic acid shifted in presence of 240 mM phosphoric acid (dashed line). B, Absorbance of increasing NcLPMO9C concentrations in 50 mM phosphate buffer, pH 6.0 (blue circles). To the same samples 1 mM ascorbic acid and 240 mM phosphoric acid were added (black circles). Color-coded dashed lines indicate the linear fit.