Redox chemistry in laccase-catalyzed oxidation of N-hydroxy compounds

Abstract

1-Hydroxybenzotriazole, violuric acid, and N-hydroxyacetanilide are three N-OH compounds capable of mediating a range of laccase-catalyzed biotransformations, such as paper pulp delignification and degradation of polycyclic hydrocarbons. The mechanism of their enzymatic oxidation was studied with seven fungal laccases. The oxidation had a bell-shaped pH-activity profile with an optimal pH ranging from 4 to 7. The oxidation rate was found to be dependent on the redox potential difference between the N-OH substrate and laccase. A laccase with a higher redox potential or an N-OH compound with a lower redox potential tended to have a higher oxidation rate. Similar to the enzymatic oxidation of phenols, phenoxazines, phenothiazines, and other redox-active compounds, an "outer-sphere" type of single-electron transfer from the substrate to laccase and proton release are speculated to be involved in the rate-limiting step for N-OH oxidation.

FIG. 1

Structures of HBT, VA, and NHA.

FIG. 2

Formal redox potentials of the N-OH compounds as a function of pH. Traces a, b, and c represent the pH dependence of E_pa of HBT, E_1/2 of VA, and E_1/2 of NHA, respectively. For HBT, there was no significant acid-base transition for its E_pa; thus, no apparent pK_a was extracted from trace a. For VA, two apparent pK_a values of 6.4 and 8.6 were extracted from trace b. For NHA, two apparent pK_a values of 3.7 and 6.3 were extracted from trace c. E_1/2 values of 0.83 and 0.91 V at pH 4 have been reported for NHA and VA, respectively, by R. Bourbonnais et al. (Oxidative Enzymes for Lignocellulose Processing, Symp. Am. Chem. Soc. 217th Nat. Meet., Anaheim, Calif., 21 to 25 March 1999).

FIG. 3

O₂ consumption in RsL-catalyzed oxidation of NHA at pH 5.5. Plotted against the initial NHA concentration [NHA] are the initial O₂ consumption rate (as the output voltage change rate) (A) and the final O₂ consumed (as the final output voltage change) (B). For graph clarity, two sets of data, obtained with 0.05 and 0.3 mM NHA, are omitted from panel B. Their symbols overlap with those for 0 and 0.33 mM NHA (shown), and their values were included in the graph fitting. In panel A, the solid curve shows the fit to the Michaelis-Menten equation {v = −V_max[NHA]/(K_m + [NHA])} with a K_m of 2.0 ± 0.5 mM and a V_max of 0.39 ± 0.03 V min⁻¹ or 0.19 ± 0.01 mM min⁻¹ (corresponding to a k_cat of 150 ± 10 min⁻¹ (mean ± standard deviation). In panel B, the horizontal broken line represents the voltage change (0.58 ± 0.03 V, averaged over the data obtained with 3.3, 5.0, and 6.7 mM NHA) corresponding to maximal O₂ consumption. Its cross point with the other broken line (voltage change, 0.5 × [NHA]; r², 0.92), obtained by fitting the data obtained with 0, 0.05, 0.30, and 0.33 mM NHA, yielded a saturating [NHA] of 1.2 mM. By dividing 1.2 mM by 0.28 mM, the dissolved [O₂] in water, we estimated an oxidation stoichiometry number of 4.1.

FIG. 4

Dependence of K_m, k_cat, and k_cat/K_m on ΔE₀. Symbols: ○, HBT; ▵, VA; ×, NHA. Correlation lines: A, log(K_m) = −1.3(ΔE₀) + 0.46 (r², 0.26); B, log(k_cat) = 4.5(ΔE₀) + 2.5 (r², 0.74); C, log(k_cat/K_m) = 5.8(ΔE₀) + 5.0 (r², 0.82). Units: A, K_m, mM; B, k_cat, min⁻¹; C, k_cat/K_m, M⁻¹ min⁻¹. ΔE₀ is reported in V. E₀ values for laccase are taken from reference . Error bars indicate standard deviations.

FIG. 5

Correlations between log(k_cat/K_m) and ΔE₀ for phenothiazines and phenoxazines. Symbols: ○, oxidation by TvL of 3 phenoxazines and 20 phenothiazines (see Materials and Methods for their formulas) (20a); ▵, oxidation of PP (E₀, 0.71 V) by TvL, RsL, MtL, StL, and MvBO at pH 5.3 to 5.5. Correlation: log(k_cat/K_m) = 5.1(▵E₀) + 6.4 (r², 0.47). Units: k_cat/K_m, M⁻¹ min⁻¹; ΔE₀, V.

FIG. 6

Correlations between log(k_cat/K_m) and ΔE₀ for laccase catalysis. Symbols: ○, oxidation of 24 phenols by 10 fungal on plant laccases (36); □, oxidation of 3 phenoxazines and 22 phenothiazines by TvL, as well as oxidation of PP by 5 fungal laccases (data from Fig. 5); ▵, oxidation of three N-OH compounds by 7 fungal laccases (data from Table 1 and Fig. 4C); +, oxidation of ABTS, K₄Fe(CN)₆ and morpholinoaniline by up to 10 fungal or plant laccases (20, 36). Other conditions: pH, 5.3 to 5.5; temperature, 20 to 25°C. Correlation: log(k_cat/K_m) = 6.4(ΔE₀) + 6.4 (r², 0.65). Units: k_cat/K_m, M⁻¹ min⁻¹; ΔE₀, V.