Enzymes

Purified T. terrestris LPMO (TtLPMO9E, previously TtGH61E) and T. auranticus (TaLPMO9A) were donated from Novozymes A/S (Denmark). The enzymes are produced by expression in a host organism and subsequently purified. T. fusca AA10 (TfLPMO10A) cloned and expressed in Escherichia coli was purchased from Nzytech Ltd (Portugal). All LPMOs were free of any residual cellulase or hemicellulose activities. Commercial cellulase mixtures Celluclast 1.5l and Novozym 188 were obtained from Novozymes A/S. The Celluclast 1.5l mixture had a protein content of 127 mg g−1, containing 62 filter paper units (FPU) per g, and a β-glucosidase activity of 15 U per g. Novozym 188 had a protein content of 220 mg g−1, containing a β-glucosidase activity of 231 U per g.

Chemicals, materials and substrates

Ascorbic acid was obtained from Sigma-Aldrich. Stock solutions of 100 mM were made in water and kept at −20 °C in the dark. Avicel microcrystalline cellulose was obtained from Sigma-Aldrich. Xyloglucan product code P-XYGLN was obtained from Megazymes Ltd, Ireland. Chlorophyllin product code C-100.000-WS-P produced by extraction of Festuca arundinacae was obtained from Chr. Hansen, Hørsholm, Denmark.

Preparation of PASC microcrystalline cellulose substrate

Avicel (microcrystalline cellulose, Sigma-Aldrich PH101) was swollen with phosphoric acid to generate PASC as described by Wood31 with a few modifications: 4 g of Avicel were suspended in 100 ml of phosphoric acid (85% w/v) at 40 °C and magnetically stirred for 1 h. The mixture was poured into 1.9 l of water at 40 °C and stirred for 1 h. The suspension was washed four times with 2 l H 2 O (MilliQ-quality), two times with 2 l of a 1% NaHCO 3 solution to reduce acidity, and then three additional times with 2 l H 2 O and stored at 4 °C. The final cellulose content of the PASC suspension was determined by enzymatic hydrolysis (24 h, 50 °C), with an enzymatic dosage of 75 FPU per g of cellulose, using Celluclast 1.5L cellulolytic enzymes and Novozym 188 in a 5:1 ratio; the released glucose was measured according to the protocol described in section ‘Enzymes’ giving an estimated cellulose content of 1.5% w/v. The average degree of polymerization (DP) of Avicel-derived PASC was determined by measuring the total number of reducing ends32 and comparing this with the total amount of monomeric glucose, giving a DP of 52.

Organosolv lignin extraction

The lignin fraction was prepared from wheat straw (Triticum aestivum L.). The straw was ball milled for 20 min and the cellulose and hemicellulose fractions were removed by hydrolytic enzymes (Celluclast 1.5L and, Novozym 188 in a 5:1 v/v) at a final dosage of 75 FPU per g of dry lignocellulose substrate for 144 h at 50 °C followed by washing with MilliQ water at a ratio of 1 l for 1 g of dry material. The amount of residual carbohydrates was <2% in the final material. Finally, the residual material was suspended in an aqueous ethanol solution (50:50 water/ethanol) at a 5:1 liquid/solid ratio and heated at 220 °C in a 1 l Parr reactor for 80 min. After heating, the lignin residue was filtered at 75 °C. Solubilized lignin was precipitated by adding water at three times the original amount and recovered by filtration. The insoluble lignin fraction was dried at 40 °C and ground with a pestle and mortar.

Preparation of chlorophyllin

A stock solution of 12% w/v (166 mM) chlorophyllin was prepared by dissolving the powder in water (MilliQ-quality). The chlorophyllin was kept in darkness while stored at 4 °C. Prior to each experiment, an aliquot of the stock solution was incubated in darkness for 2 h at room temperature.

Preparation of cyanobacterial thylakoid suspensions

Thylakoid suspensions, containing light-harvesting antennae (phycobilisomes) and thylakoid membranes, were prepared from the cyanobacterium Synechococcus sp. PCC 7002 (referred to as Synechococcus) grown in medium A containing 2 g NaNO 3 per l as previously described33. Synechococcus cells were harvested in 50 ml volumes by centrifugation (5,000g for 5 min) and subsequently resuspended in 1 ml thylakoid washing buffer (pH 6.35) as described34. The resolved pellet was transferred to a microfuge tube containing 500 μl glass beads (glass beads for cell disruption, 0.1–0.25 mm diameter, Retsch Technology GMBH, Haan, Germany) followed by cell disruptive sonication (Amplitude 50, 3 min processing time, 5 s on/off cycle). The cell extract was then centrifuged (12,000g, 4 °C, 20 min) and the pellet, containing unbroken cells and cell walls, was discarded. An additional centrifugation (40,000g, 4 °C, 30 min) separated the light-harvesting antennae (phycobilisomes, supernatant) from the thylakoid membranes (pellet). Absorption spectra of the thylakoid suspensions in the supernatant were performed with a UV1800 spectrophotometer (Shimadzu, Kyoto, Japan) and the chlorophyll a content was calculated as described35. The thylakoid suspensions were then used to conduct the light-induced electron-transfer experiments with an average concentration of 0.21±0.06 mg Chl per ml. Putative contaminations derived from cytoplasmic membranes in the thylakoid suspension have no impact on light-induced electron transfer to LPMOs, this was tested with disrupted E. coli K-12 cells

Preparation of plant thylakoid membranes

Plant thylakoid membranes were extracted from A. thaliana (L.) Heynh. Ecotype Columbia. Plants were grown in compost in a controlled environment Arabidopsis chambers (Percival AR-60 I, Boone, IA) at a photosynthetic flux of 130–150 μmol of photons per m2 per s, 20 °C and 70% humidity. Leaves from ∼25 plants were pooled and homogenized using a blender fitted with razor blades in ice cold buffer containing 20 mM Tricine (pH 7.5), 10 mM NaCl, 5 mM MgCl 2 , 0.4 M sucrose, 5 mg ml−1 bovine serum albumin (BSA) and 100 mM sodium ascorbate. The homogenate was immediately filtered through two layers of nylon mesh (31 μm pore size), after which the filtrate was centrifuged (6,000g, 4 °C, 15 min). The pellet was resuspended in 5 mM Tricine (pH 7.9) to lyse the chloroplasts. Following lysis, the thylakoids were collected by centrifugation (17,200g, 4 °C, 10 min). The pellet was resuspended in a small volume of homogenization buffer without ascorbate and BSA but with 20% glycerol (v/v). Total chlorophyll (Chl) and Chl a/b ratio were determined in 80% acetone according to the study by Lichtenthaler36. The final concentration of the thylakoid membranes applied in the light-induced electron-transfer experiments was 3.57 mg Chl per ml (Chl a/b ratio=3.0).

Elemental analysis inductively coupled plasma (ICP)

Chlorophyllin and thylakoid suspensions were analysed for the presence of metals on an Aurora Elite ICP-MS system from Bruker. Samples of chlorophyllin 12% (w/w) and extracted thylakoid suspensions 2% (w/w) were diluted to 500–1,000 p.p.b. dry matter in 1% nitric acid. The samples were analysed for 24Mg, 25Mg, 26Mg 63Cu, 65Cu, 66Zn, 67Zn and 68Zn. Chlorophyllin contained 0.41% Mg, 2.0% Cu and 0.05% Zn relative to total dry matter. Extracted thylakoid suspensions contained 1.8% Mg, 0.007% Cu and 0.0007% Zn relative to total dry matter.

Quantification of glucose and cellobiose by HPLC

The quantification of D-glucose and D-cellobiose was done using an Ultimate 3000 high-performance liquid chromatography (HPLC, Dionex, Germering, Germany) equipped with refractive index detector (Shodex, Japan) and ultraviolet detector at 210 nm (Dionex). The separation was performed in a Phenomenex Rezex ROA column at 80 °C with 5 mM H 2 SO 4 as eluent at a flow rate of 0.8 ml min−1.

Measurement of oligosaccharide and gluconic acid by HPAEC

The samples were prepared as follows: 200 μl were centrifuged at 14,000g for 2 min and 100 μl of the supernatant was inserted in the HPLC conical vial, without any further light exposure (wrapped in aluminium foil). HPAEC was run on an ICS5000 system (two different machines available in the laboratory), equipped with a PAD detector (Dionex, Sunnyvale, CA, USA) with a CarboPac PA1 column (2 × 50 mm guard column followed by a 2 × 250 mm analytical column) and operated at a flow of 0.25 ml min−1, at 30 °C. Chromatographic analysis of aldonic acids separation was conducted as described by Westering et al.37. The elution involved a linear gradient from 100% A:0% B to 90% A:10% B (10 min), followed by an exponential gradient to 70% A:30% B (15 min) and last an exponential gradient to 100% B (5 min). After that a linear gradient was applied for 15 min at the initial conditions 100% A:0% B (eluent A=0.1 M NaOH, B=0.1 M NaOH and 1 M NaOAc)37. The peaks of the cellulose oligomers in the chromatograms are assigned according to Westereng et al.37. The chromatograms shown in the main paper, and in Supplementary Figs 1–7, are average of three independent experiments.

Quantification of cellulose oxidation for TtLPMO9E

To quantify the total oxidation of cellulose (as shown in Fig. 1c), the residual PASC together with the supernatant containing oligosaccharides were digested with a commercial cellulase mixture, Celluclast supplemented with beta-glucosidase Novozym 188 in a 5:1 v/v ratio (both lacking LPMO activity) and applied based on Celluclast at 75 f.p.u. per g cellulose for 5 h at 50 °C in darkness. These conditions ensured complete hydrolysation of the PASC material. The LPMO reaction was stopped prior to cellulases treatment by boiling the vials/samples for 10 min. The hydrolysates were then analysed by HPLC to quantify the D-glucose and in the HPAEC for quantification of gluconic acid (marker of C1 oxidizing activity of LPMO). A minor C4 component (annotated C4-oxidized after minute 24) appeared in the oxidized products; it was impossible to quantify the amount of the monomeric 4-keto aldose sugar (C4-oxidized glucose) as due to tautomerization there are no standards available.

Standard conditions for enzymatic reactions and LIET

The light-induced electron-transfer (LIET) system was composed of PASC incubated with an LPMO enzyme, a light-absorbing component (chlorophyllin, cyanobacterial thylakoid suspensions or plant thylakoid membranes) and a reductant, that is, ascorbic acid. All experiments presented in the paper, as well as in the Supplementary Information were conducted in triplicates, and repeated on two different ICS5000 HAPEC systems available in the laboratory.

The standard reaction mixture composition for PASC oxidation reactions was: 0.75% w/v PASC, 2 mM ascorbic acid, 100 mM of citrate-phosphate buffer (pH 6.3) and 0.05 mg ml−1 LPMO in a 200 μl reaction volume. For the TtLPMO9E with an estimated molecular weight of 22.5 KDa this equals 2.2 nmol of added enzyme. Chlorophyllin was added in a 1:100 dilution out of a 12% stock solution thus a final concentration of 1.6 mM. Freshly prepared cyanobacterial thylakoid suspensions and plant thylakoid membranes were diluted into the reaction mixture 1:2 and 1:20, respectively. The described composition applies for reactions using the AA9 enzymes (TtLPMO9E and TaLPMO9A) and is referred as standard conditions in the rest of the Methods section. For reactions with the AA10 enzyme (TfLPMO10A), a phosphate buffer at pH 7.8 (20 mM) was used and 0.20 mg ml−1 of the enzyme preparation was used.

The standard conditions for the light-induced electron-transfer system for cellulose oxidation are defined as: 3 h incubation time, irradiance intensity for sunlight was 150–200 μmol photons per m2 per s, for blue, red and green light sources 150 μmol of photons per m2 per s (custom made LEDs for blue light 440 nm, red light 625 nm and green light 528 nm, with a spectral width 18 nm, 18 nm and 33 nm respectively). The irradiance intensities reported were measured at the bottom of the closed 2 ml Eppendorf test tubes, and the light probe (a Spherical Micro Quantum Sensor US-SQS/Lund) was submerged into the reaction mixture to account for light-scattering effects caused by PASC cellulose or other insoluble particles. Incubations were carried out in 2 ml Eppendorf polypropylene microfuge tubes in a thermomixer at 1,000 r.p.m. and 50 °C (Eppendorf thermomixer, Eppendorf, Hamburg, Germany). The thermomixer was modified to carry a transparent microfuge tube rack allowing light penetration through the walls of the microfuge tubes. All experimental preparations were carried out in dim green light at 0 μmol of photons per m2 per s (Phillips TL-D 36 W colour green Lumen 3600, 540 nm). The degree of PASC oxidation was determined by quantification of oxidized glucose units being the gluconic acid. Optimization of reaction conditions in terms of time of incubation and reactant dosages are shown in Supplementary Fig. 8.

In addition to the standard reactions described above, multiple controls were performed as follows: darkness controls were wrapped in aluminium foil; LPMO activity controls were without photopigments or without light exposure and were incubated with ascorbic acid and PASC; non-enzymatic oxidation controls of chlorophyllin, thylakoid suspensions, lignin and ascorbic acid were individually incubated with PASC and without LPMO enzymes under standard conditions (see above) to assess the possibility of spontaneous cellulose oxidation caused by light exposure (Fenton-type reactions).

Sequential darkness or sunlight exposure

To verify the role of light as an inducer of cellulose oxidation via chlorophyllin, darkness and sunlight were sequentially applied to the same samples (results in Fig. 1d). The experiment was performed using the standard conditions for light-induced electron transfer (see above). The reaction mixture contained PASC, chlorophyllin, ascorbic acid and TtLPMO9E with a lower dosage, using only 4 μg (instead of 10 μg) for a lower level of product release. The reaction mixtures were exposed to darkness for 2 h followed by sunlight exposure for 5 min. This cycle was subsequently repeated. After each change in light/darkness exposure, an aliquot was removed from the vials for further analysis. All aliquots were analysed by high-performance anion exchange chromatography (HPAEC) to measure the release of oligosaccharides and additionally treated with cellulase to quantify the monomeric glucose and gluconic acid (see above). An LPMO activity control, applying the same LPMO dosage was incubated for 24 h and afterwards treated with cellulase to quantify the monomeric glucose and gluconic acid (see above).

Oxygen consumption measurements

The oxygen consumption was measured with a Chlorolab 2 System (Hansatech, England), with an oxygen sensor mounted at the bottom of a sealed reaction chamber. The 1 ml reaction chamber was stirred by a magnet and was equipped with a window for light exposure. Reactants could be added via a capillary cannula at the top, and no oxygen exchange with the external environment was observed. The measurements are given as oxygen concentration in μmol per ml. White LED light (4100 K) at 150 μmol photons per m2 per s was applied. The reactants were added at the beginning according to the dosages explained in the standard conditions, but at a lower temperature of 25 °C. Note that there is no headspace in the reaction chamber, why much less oxygen is available compared with the conditions described in standard conditions for enzymatic reactions. The combinations tested were: complete photosystem: LPMO (TtLPMO9E)+chlorophyllin+Asc+PASC; the negative control experiments: chlorophyllin+Asc+PASC (no enzyme present); and the LPMO+Asc+PASC (no pigment present). From 0 to 900 s these reactions were carried out in darkness, while from 900 to 2,400 s white light was supplied (results shown in Fig. 2a).

The complete photosystem (LPMO+chlorophyllin+Asc+PASC) was measured with alternating cycles of light/darkness without or with the pigment. The conditions were identical to standard (see above) with the difference that TtLPMO9E was dosed at only 0.01 mg ml−1 to lower the rate of oxygen consumption, and a temperature of 25 °C. TtLPMO9E, ascorbic acid and PASC were first added. After five cycles of light and darkness (2 min each), chlorophyllin was added through the cannula, and three cycles of light and darkness were alternated (results shown in Fig. 2b).

Lignin as reductant

Ascorbic acid, generally used as a reductant in the standard conditions for the enzymatic hydrolysis, was replaced with organosolv-extracted lignin to investigate its putative reducing capacity. The organosolv lignin was suspended in citrate-phosphate buffer (pH 6.3) and was added to a final concentration of 5 mg ml−1 (equal to a molar concentration of 25 mM based on lignin monomers) to the reaction mixture and incubated under standard conditions (see above) with TtLPMO9E. Control experiments were performed as described above, replacing ascorbic acid with lignin.

Light-induced oxidation of xyloglucans

Xyloglucan 1% w/w was used as a substrate, replacing PASC and incubated with chlorophyllin, ascorbic acid and TtLPMO9E, the others parameters were according to standard conditions (see above). A release of oligosaccharides was detected using HAPEC. The observed pattern was a mixture of cello-oligosaccharides and xylogluco-oligosaccharides, which could not be fully separated, as seen for other published works19,20, therefore the peaks are separated into non-oxidized and oxidized products only.

Light-induced oxidation of crystalline cellulose

Avicel was used as a cellulose substrate at 1% w/w instead of PASC and all other conditions were same as standard conditions (see above).

Protein structure modelling

The PDB structures from the RCSB PDB Protein data bank were adapted for the HARLEM Molecular Modelling Package program (Kurnikov et al, available at http://harlem.chem.cmu.edu/). The PDB structures were (after adding hydrogens) analysed in HARLEM for possible LRET pathways from the surface to the metal ion21.