Laccases as palladium oxidases

Combining a palladium-based complex with a laccase allows the oxidation of an alcohol substrate at room temperature and atmospheric pressure.


Standard enzyme activity assay.
Laccase activity was assayed at 30°C using syringaldazine (SGZ) as substrate. Oxidation of SGZ was detected by following the absorbance at 525 nm ( 525 nm = 6.5x10 4 M -1 .cm -1 ) during 2 min using a spectrophotometer (Carry 50, Varian). The reaction mixture (1 mL) contained 10 µL of appropriately diluted enzyme sample, 960 µL of acetate buffer (100 mM pH 5.7) and 30 µL of 0.8 mg/mL SGZ in MeOH at 30°C. The SGZ was added to initiate the reaction. One unit (U) of laccase was defined as one micromole of substrate oxidized per minute in these described conditions.

Bleaching experiments
UV/VIS. Experiments were performed on a VARIAN Cary 50 spectrophotometer. With complex 1: 209 M laccase LAC3, 2 mM complex 1 in the absence or presence of 200 mM veratryl alcohol in acetate buffer (33 mM pH 5.7) at 25 °C. With complex 4: 70 M laccase and 0 to 700 M of complex 4 in acetate buffer (33 mM pH 5.7) at 25 °C under anaerobic conditions (Fig. SI1A). Kinetics of reduction were followed at 610 nm. ESR. Spectra were obtained using a BRUKER EMX9/2.7 spectrometer equipped with a B-VT2000 digital temperature controller (100-400 K). With complex 1: 209 µM LAC3, 10 equivalents of complex 1 in the absence or presence of veratryl alcohol 200 mM in 33 mM acetate buffer pH 5.7. With complex 4: 180 µM of laccase, 10 equivalents of complex 4 in 33 mM acetate buffer pH 5.7. Samples were placed in J. Young valved ESR tubes under inert atmosphere and analysed as frozen solutions (115 K, Fig. SI1B).

Dioxygen consumption experiments.
Dioxygen consumption was measured by potentiometry using a model 781 oxygen meter (Strathkelvin Instruments) with a micro Clark electrode fitted to a temperature controlled glass chamber (1.0 ml). A solution of 50 µM complex 1, 50 mM veratryl adehyde was placed in the chamber. A decrease in dioxygen concentration was followed (250µM initial concentration at 25 °C) then laccase (5 µM) was added to the mixture followed by NaN 3 (100 µM).

Oxidation of veratryl alcohol assay.
In a typical experiment, oxidation of veratryl alcohol was performed in 15 mL tube at 25 °C with gentle constant shaking. Freshly prepared complex solution (0 to 100 µM based on palladium) was incubated with veratryl alcohol (0 to 200 mM) in 500 µL 33 mM acetate buffer pH 5.7 for 24 hours. When laccase LAC3 was present, 0 to 5 µM (based on T1 extinction coefficient  610nm = 5600 M -1 .cm -1 ) were added to the mixture. Samples were then applied onto a C18 nucleosil column (300 A, 250*4.6 mm) after extraction with an ethylacetate solution containing 1 mM benzophenone as a standard for quantification, samples were analysed by High Performance Liquid Chromatography (Waters 2695 equipped with a photodiode array detector). The aldehyde concentration was determined spectrophotometrically at 310 nm from a standard curve performed with commercial aldehyde solutions.

Influence of LAC3 and NaN 3 on product formation.
Oxidation of veratryl alcohol was performed in 2 mL 1 cm path-lengh UV-Visible cell with gentle constant shaking on a Varian Cary 50 bio equipped with a thermostat and followed at 310 nm (Veratryl aldehyde : 310 nm = 9300 M -1 .cm -1 ). In a typical experiment, 50 µM complex 1 with 20 mM substrate in presence of LAC3 (0 to 10 µM) in 2 mL of 33 mM acetate buffer pH 5.7 at 25 °C. For inhibition studies, 100 µM NaN 3 were added to the mixture. Vi (initial velocities) were extracted with a linear fitting of the curve obtained after 150 min of reaction.

Electrophoresis experiments
Standard sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) (7.5 % acrylamide) were used for the separation of denatured protein samples (10 min at 100°C in Laemmli loading buffer) 5 . 20 µL of each sample were loaded on gel after the thermal denaturation under reducing conditions. The gels were then stained with coomassie and decolorized with a EtOH/Acetic acid/ H 2 O (4/1/5) solution.

Influence of the reaction time on catalytic efficiencies
Oxidation of veratryl alcohol was performed in 15 mL tube at 25 °C with gentle constant shaking. 50 µM of freshly prepared complex solution was incubated with 20 mM veratryl alcohol in 500 µL 33 mM acetate buffer pH 5.7 for 24, 44 and 72 hours . When laccase LAC3 was present, 0 to 5 µM were added to the mixture.

A.
B. C.

Influence of different oxidation agents on alcohol oxidation
Quinones are well known re-oxidation agents for PD complexes. For the sake of comparison with LAC3, we checked the activity of the Pd complex (1) in the presence of an equimolar amount of benzoquinone ± 1 equivalent of CuSO 4 . Results are presented Fig. SI7. Figure SI7. Influence of different oxidation agents on alcohol oxidation. Reactions were performed in the presence of ±5 µM LAC3, ±50 µM complex 1, ± 50 µM benzoquinone (Bz), ± CuSO 4 (Cu) and 100 mM final substrate concentration in 33 mM acetate buffer pH 5.7 at 25 °C in 15 mL tubes with rotary shaking for 24h. Average of 3 sets of independent reactions. The relative activity (%) is set to 100 for complex 1.

Influence of catalyst load as function of time
The conversion of veratryl alcohol (20 mM veratryl alcohol at 22 °C in 33 mM acetate pH 5.7) into veratryl aldehyde was followed as function of time and catalyst load (from 0.125 to 2.5%). Values (average of three independent values) are presented in Table SI1.