Demonstration of Lignin-to-Peroxidase Direct Electron Transfer: A TRANSIENT-STATE KINETICS, DIRECTED MUTAGENESIS, EPR, AND NMR STUDY

Abstract

Versatile peroxidase (VP) is a high redox-potential peroxidase of biotechnological interest that is able to oxidize phenolic and non-phenolic aromatics, Mn(2+), and different dyes. The ability of VP from Pleurotus eryngii to oxidize water-soluble lignins (softwood and hardwood lignosulfonates) is demonstrated here by a combination of directed mutagenesis and spectroscopic techniques, among others. In addition, direct electron transfer between the peroxidase and the lignin macromolecule was kinetically characterized using stopped-flow spectrophotometry. VP variants were used to show that this reaction strongly depends on the presence of a solvent-exposed tryptophan residue (Trp-164). Moreover, the tryptophanyl radical detected by EPR spectroscopy of H2O2-activated VP (being absent from the W164S variant) was identified as catalytically active because it was reduced during lignosulfonate oxidation, resulting in the appearance of a lignin radical. The decrease of lignin fluorescence (excitation at 355 nm/emission at 400 nm) during VP treatment under steady-state conditions was accompanied by a decrease of the lignin (aromatic nuclei and side chains) signals in one-dimensional and two-dimensional NMR spectra, confirming the ligninolytic capabilities of the enzyme. Simultaneously, size-exclusion chromatography showed an increase of the molecular mass of the modified residual lignin, especially for the (low molecular mass) hardwood lignosulfonate, revealing that the oxidation products tend to recondense during the VP treatment. Finally, mutagenesis of selected residues neighboring Trp-164 resulted in improved apparent second-order rate constants for lignosulfonate reactions, revealing that changes in its protein environment (modifying the net negative charge and/or substrate accessibility/binding) can modulate the reactivity of the catalytic tryptophan.

Keywords: HSQC NMR; electron paramagnetic resonance (EPR); enzyme kinetics; lignin degradation; lignosulfonate; nuclear magnetic resonance (NMR); peroxidase; transient-state kinetics; tryptophanyl radical; versatile peroxidase.

FIGURE 1.

Environment of the exposed catalytic tryptophan acting as starting point for LRET to heme in VP. Trp-164, 10 neighbor residues (those changed by directed mutagenesis in bold), heme (all as Corey-Pauling-Koltun-colored sticks), and semitransparent protein surface (blue, except for the above tryptophan, in yellow, and the neighbor residues, in Corey-Pauling-Koltun colors) are shown

FIGURE 2.

Tentative structure for sulfonated softwood lignin (22) and simple model compounds. a, structure of softwood lignosulfonate as Ca²⁺ salt (LS, lignosulfonate chains; MeO, methoxyl group). b and c, formulae of creosol (b), and α-sulfonated creosol (c). The basic phenylpropanoid unit is shown (box), and carbon labeling is indicated. See Fig. 8e for the main structures identified in softwood (P. abies) and hardwood (E. grandis) lignosulfonates

FIGURE 3.

Kinetics of reduction of CI (a and c) and CII (b and d) of native VP (●) and its W164S (○) and R257A/A260F (■) variants by softwood (a and b) and hardwood (c and d) lignosulfonates. Stopped-flow reactions were carried out at 25 °C in 0.1 m tartrate (pH 3). The lignosulfonate concentrations in Figs. 3–5 refer to the basic phenylpropanoid unit, as explained under “Experimental Procedures.” Means and 95% confidence limits are shown. Insets show the R257A/A260F kinetic curves for a smaller concentration range. Error bars indicate means ± S.E.

FIGURE 4.

Kinetics of VP CI (a) and CII (b) reduction by creosol (black circles) and the corresponding sulfonate derivative (white circles). Stopped-flow reactions were carried out at 25 °C in 0.1 m tartrate (pH 3). Means and 95% confidence limits are shown. Error bars indicate means ± S.E.

FIGURE 5.

EPR spectra of the reactions of VP with H₂O₂ (at molar ratio 1:8), and of VP with H₂O₂ and softwood lignosulfonate at two different molar ratios (1:8:4 or 1:8:12). All spectra were recorded at 40 K, 9.394 GHz, 1-milliwatt microwave power, and 0.2 mT modulation amplitude, a few seconds after mixing. Intensity-normalized spectra are shown (integration values for the tryptophanyl and lignin radical signals in the original spectra are provided under “Results”).

FIGURE 6.

Relative fluorescence of softwood (a) and hardwood (b) lignosulfonates during 24-h treatment with VP and H₂O₂ (black bars) and the corresponding controls without enzyme (white bars). Changes of lignosulfonate (12 g/liter⁻¹) fluorescence during treatment with VP (1.3 μm) and H₂O₂ (12.5 mm) in 50 mm phosphate (pH 5) were monitored (excitation at 355 nm/emission at 400 nm) after different time periods (3, 12, and 24 h). Means and 95% confidence limits are shown. Error bars indicate means ± S.E.

FIGURE 7.

Molecular mass distribution of VP-treated and control softwood (a) and hardwood (b) lignosulfonates, and sulfonated polystyrene standards (c). Lignosulfonate samples (12 g/liter⁻¹) after a 24-h treatment with VP (1.2 μm) and H₂O₂ (9.5 mm) (red lines), and the corresponding controls without enzyme (green lines), were analyzed in a Superdex-75 column using 0.15 m NaOH as eluent (0.5 ml min⁻¹) and detection at 280 nm. Sulfonated polystyrenes (Mp 78,400, 29,500, 10,200, and 4,210 Da, from left to right) were used as molecular mass standards (arrow shows the blue dextran elution volume) (c).

FIGURE 8.

HSQC spectra of softwood (a–c) and hardwood (d–f) lignosulfonates after 3-h (b and e) and 24-h (c and f) treatment with VP/H₂O₂, as compared with control without enzyme (a and d), and formulae of the main structures identified (g). Signals correspond to ¹³C-¹H correlations at the different positions of lignin normal/α-oxidized/α-sulfonated syringyl units (red signals), guaiacyl units (green signals), α-sulfonated/non-sulfonated side chains in β-O-4′ (blue signals), phenylcoumaran (cyan signals), and pinoresinol (purple signals) substructures, and methoxyls (orange signals) (gray, unassigned signals). Signals of β-O-4′ substructures with a second guaiacyl or syringyl unit could be identified. The same amount of sample (40 mg before treatment) and DMSO-d₆ (0.75 ml) were used for all the spectra in Figs. 8 and 9, which were normalized to the same intensity of the DMSO signal (not shown) for comparison. List of signals (δ_C/δ_H ppm): 53.2/3.46, C_β/H_β in phenylcoumarans (B_β); 53.4/3.00, C_β/H_β in resinols (C_β); 55.5/3.66, C/H in methoxyls (MeO); 59.4/3.4 and 3.72, C_γ-H_γ in β-O-4′ (A_γ); 61.1/4.00, C_γ-H_γ in sulfonated β-O-4′ (A_γ); 65.6/3.93, C_α/H_α in sulfonated β-O-4′ linked to a G unit (A_α(G)); 67.2/4.02, C_α/H_α in sulfonated β-O-4′ linked to an S unit (A_α(S)); 70.8/4.16 and 3.77, C_γ-H_γ in β-β′ resinols (C_γ); 71.1/4.72, C_α/H_α in β-O-4′ linked to a G unit (A_α(G)); 71.5/4.85, C_α/H_α in β-O-4′ linked to an S unit (A_α(S)); 79.3/4.91, C_β/H_β in sulfonated β-O-4′ linked to a G unit (A_β(G)); 80.9/4.67, C_β/H_β in sulfonated β-O-4′ linked to an S unit (A_β(S)); 83.3/4.24, C_β/H_β in β-O-4′ linked to a G unit (A_β(G)); 84.9/4.59, C_α/H_α in β-β′ resinols (C_α); 85.7/4.08, C_β/H_β in β-O-4′ linked to an S unit (A_β(S)); 86.7/5.41, C_α/H_α in phenylcoumarans (B_α); 103.8/6.68, C₂/H₂ and C₆/H₆ in syringyl units (S_2,6); 106.2/7.29, C₂/H₂ and C₆/H₆ in α-oxidized syringyl units (S′_2,6); 108.0/6.68, C₂/H₂ and C₆/H₆ in sulfonated syringyl units (S_2,6); 114.0/6.60 and 114.3/6.87, C₂/H₂ and C₅/H₅ in guaiacyl units (G₂/G₅); and 122.8/6.75, C₆/H₆ in guaiacyl units (G₆) (minor, and largely overlapping, signals of C₂/H₂, C₅/H₅, and C₆/H₆ correlations in non-sulfonated guaiacyl units would appear at 110.7/6.93, 114.2/6.65, and 118.6/6.79 ppm, respectively; not shown).

FIGURE 9.

¹³C NMR spectra of softwood (a and b) and hardwood (c and d) lignosulfonates after a 24-h treatment with VP/H₂O₂ (b and d), as compared with control without enzyme (a and c). Signals of protonated (G₂, G₅, G₆, and S/S_2,6) and quaternary (G₁, G₃, and G₄, and S₁, S₃, S₄, and S₅) carbons in guaiacyl and syringyl lignin units; α/β carbons in β-O-4′ linked sulfonated (A_α and A_β) and non-sulfonated (A_α and A_β) lignin side chains; and methoxyls (MeO) are shown, together with a carboxyl (R-COOH) signal. Two sharp extra signals, at 59.2 and 61.0 ppm (one of them also observed in the HSQC spectra), most probably come from the buffer in lignosulfonate dialysis (before use), and the other in the treated samples remained to be assigned. List of quaternary carbon signals (δ_C ppm): 134, C₁ in guaiacyl units (G₁) and C_1,4 in syringyl units (S_1,4); 147, C_3,4 in guaiacyl units (G_3,4); and 152, C_3,5 in syringyl units (S_3,5) (see Fig. 8 legend for δ_C of protonated carbons).

FIGURE 10.

VP catalytic cycle. Shown is a scheme for VP catalytic cycle displaying resting state (Fe³⁺) activation by H₂O₂ and lignin oxidation by a tryptophanyl radical (VP-I_B and VP-II_B) formed by one electron transfer from Trp-164 to VP-I_A (Fe^IV=O·porphyrinyl radical, P^•+, complex) and VP-II_A (Fe^IV=O) heme. In contrast, Mn²⁺ is directly oxidized by VP-I_A and VP-II_A. Other VP substrates, like phenols (including the lignin phenolic units) and dyes, can be oxidized both at the heme access channel (by VP-I_A and VP-II_A) and at the catalytic tryptophan (by VP-I_B and VP-II_B) (38) (not shown for simplicity). The above porphyrinyl radical was experimentally observed in the EPR spectrum of the peroxide-activated W164S variant (at 9 K), whereas the tryptophanyl radical was observed in VP spectra acquired at 40 K. The transient state (apparent second-order) rate constants for reactions with sulfonated lignins can be overestimated because some reaction is also produced at the heme channel (by VP-I_A and VP-II_A), probably involving the minor phenolic units in lignin, as shown in Table 1 (W164S variant). The H₂O₂ and Mn²⁺ rate constants are taken from previous studies (16, 46). No constants are provided for the pass of VP-I_A and VP-II_A to VP-I_B and VP-II_B, respectively, because electron deficiency is shared between the two redox centers (18).