Fig. 3: Probing the role of reactive oxygen species in the light/lignin/LPMO system.

The graphs show time-courses for the formation of soluble oxidized products (a, c) and the corresponding apparent catalytic rates (b, d) for reactions with Avicel (10 g L⁻¹), ScAA10C (0.5 µM), kraft lignin (0.9 g L⁻¹), and light-exposure in the presence of varying amounts of horseradish peroxidase (HRP) (a, b) and superoxide dismutase (SOD) (c, d). The varying colors in panels a and c indicate different time points of the reaction, as explained in the graphs. The rates shown in b were derived from linear regression analysis using all three time points in a with R² > 0.99 for all reactions with 0, 19.3, 193 nM HRP except for one replicate with 193 nM with R² > 0.93. For the reactions with 1930 nM HRP the product levels were very low and showed larger variability as these levels were close to the detection limit of the analytical method. The rates in d were derived using linear regression analysis for all time points displayed in c and all reactions gave progress curves with R² > 0.99. All reactions were carried out in sodium phosphate buffer (50 mM, pH 7.0) at 40 °C, under magnetic stirring and exposed to visible light (I = 10% I_max, ~16.8 W cm⁻²). Before quantification of soluble oxidized products, solubilized cello-oligosaccharides were hydrolyzed by TfCel6A to convert LPMO products with varying degree of polymerization (DP) to a mixture of DP 2 and 3 [GlcGlc1A, (Glc)2Glc1A], the amounts of which were summed up to yield the concentration of oxidized sites. The data presented are mean values derived from three (a, b) or two (c, d) independent experiments; error bars show ±s.d. (a, b; n = 3).