Light-driven oxidation of polysaccharides by photosynthetic pigments and a metalloenzyme

Oxidative processes are essential for the degradation of plant biomass. A class of powerful and widely distributed oxidative enzymes, the lytic polysaccharide monooxygenases (LPMOs), oxidize the most recalcitrant polysaccharides and require extracellular electron donors. Here we investigated the effect of using excited photosynthetic pigments as electron donors. LPMOs combined with pigments and reducing agents were exposed to light, which resulted in a never before seen 100-fold increase in catalytic activity. In addition, LPMO substrate specificity was broadened to include both cellulose and hemicellulose. LPMO enzymes and pigment derivatives common in the environment of plant-degrading organisms thus form a highly reactive and stable light-driven system increasing the turnover rate and versatility of LPMOs. This light-driven system may find applications in biotechnology and chemical processing.

His87 through His86 to Cu(II) through 12 covalent bonds equivalent to a tunneling length of 16.8 Å with a calculated pathway coupling decay value of 2.2 10 -3 from His87 NE2 to Cu(II). His87 is sitting on the side of LPMO and is thus free of cellulose that is binding to LPMO during catalysis.
In another structure of the same enzyme soaked in 10 mM Cu(NO 3 ) 2 (PDB 3ZUD) a Cu(II) ion is found coordinating to this His87 supporting that it is a solvent exposed amino acid. 5 A search conducted on other structures in the AA9 family revealed that similar pathways could be found.

Light-induced effects of the individual assay components
Detailed HPAEC chromatograms of PASC oxidation by T. terrestris LPMO or thylakoids or chlorophyllin exposed to sunlight and compared to a control containing only LPMO and ascorbic acid are shown in Supplementary Figures 5 and 6.
The ability of pigments to harvest light and transfer electrons upon excitation was first observed having LPMO incubated with pigments only (thylakoids or chlorophyllin, Supplementary Figure 5 and 6 respectively) and PASC, without reductant (ascorbic acid). The reaction mixtures were incubated in sunlight or in darkness, showing PASC oxidation only when the mixture was exposed to sunlight (yellow line). Furthermore, as negative control, LPMO was incubated with PASC and exposed to sunlight.
Supplementary Figure 6 shows, as a positive control experiment, the activity of LPMO when incubated with ascorbic acid and PASC, exposed to sunlight (red line); same experiment conducted in darkness resulted in the same level of PASC oxidation and profile of oxidized products (black line). There is no indication of a light-induced response by incubation of LPMO and ascorbic acid.
For the sunlight exposed LPMO, no PASC oxidation products were detected, neither was there any reaction for LPMO and pigments in darkness (Supplementary Figure 5 and 6, dotted and green line, respectively). The incubation parameters and reactant dosages were chosen accordingly to the standard conditions described in Methods section of the main text.
The overall amount of PASC oxidation with the pigments and enzyme only is about 10 times lower than what is observed for the full light-induced system including a reductant (Fig. 1a,b main paper).
Without the reductant the pigments are degraded by photobleaching. The distribution between oxidized and non-oxidized cello-oligomers is different for thylakoids and chlorophyllin, however, this difference disappears when the reductant is added and the artificial photosystem is complete, see Figure 1a and 1b. Further experimental details are described in Methods section of the main text.

Optimization of reaction parameters
To test that the reaction was not rate-limited, the rate of oxygen consumption for the sequential experiment having different amounts of initial dissolved oxygen (respectively 0.31, 0.29 and 0.27 µmol mL -1 ), was measured to be constant at 0.12±0.005 nmol mL -1 s -1 , see figure 2b main text.
Below is described in details the optimization of the reaction parameters evaluated in terms of final amount of cellulose oxidation in percentage of the initial amount of substrate. Dioxygen is needed for LPMO catalysis, and if not available in sufficient amount it may be limiting for the oxidation of the cellulose. In the headspace and liquid a total of 14.3 μmol dioxygen is present at 50 o C. The measured oxidation of 10% of the PASC (Fig. 1c) requires 0.56 μmol of dioxygen, thus oxygen is in excess compared to the observed level of oxidation.
The effect of increased oxygen concentration was tested by supplementing pure dioxygen gas in the 1.8 mL head space of the reaction vial just before the start of the reaction. As shown in Supplementary Figure 8a after 3 hours, the level of oxidation was on average 15% higher when 5 times more oxygen (blue bar) was supplied compared to the standard condition with atmospheric air (green bar). However, the oligosaccharide amounts were identical when the incubation was prolonged to 24 hours (data not shown). This shows that the dioxygen level present in the samples was sufficient for the assays performed. This result further supports that a putative oxygen production by thylakoids upon light exposure was not the cause of the increased level of oxidation of cellulose observed in Fig.1a.
Different dosages of chlorophyllin were tested with 0.32, 1.6 and 8 mM chlorophyllin in the final reaction volume. 3 hours of sunlight exposure and standard conditions were applied as described in Methods section of the main text. Less than 5% increase in oxidized cellulose was observed comparing 0.32 to 1.6 mM and a 15% higher level of oxidized cellulose was observed comparing 1.6 mM to 8 mM of chlorophyllin. 1.6 mM was therefore chosen as the standard concentration of chlorophyllin.
Ascorbic acid was applied in three different amounts: 0.4, 2 and 10 mM together with chlorophyllin at 1.6 mM and TtLPMO9E. All samples were exposed to sunlight for 3 hours. The increase of oligosaccharides production was proportionally correlated with the ascorbic acid molarity, when comparing 0.4 to 2 mM of ascorbic acid. However, only a 20% increase of oxidation products was achieved by increasing the amount of ascorbic acid from 2 mM to 10 mM. Thus 2 mM was chosen as the standard dosage of ascorbic acid.
The light-induced electron transfer based on chlorophyllin and the standard conditions (described in Methods section of the main text) was performed for 10 minutes, 3 and 24 hours. In supplementary figure 8d shows that within 10 minutes 2.3% of cellulose was, whereas 10.2 % and 12.3% of cellulose oxidation within 3 and 24 hours respectively.