Thermodynamics and Kinetics of Electron Transfer of Electrode-Immobilized Small Laccase from Streptomyces coelicolor

Abstract

The thermodynamic and kinetic properties for heterogeneous electron transfer (ET) were measured for the electrode-immobilized small laccase (SLAC) from Streptomyces coelicolor subjected to different electrostatic and covalent protein-electrode linkages, using cyclic voltammetry. Once immobilized electrostatically onto a gold electrode using mixed carboxyl- and hydroxy-terminated alkane-thiolate SAMs or covalently exploiting the same SAM subjected to N-hydroxysuccinimide+1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (NHS-EDC) chemistry, the SLAC-electrode electron flow occurs through the T1 center. The E°' values (from +0.2 to +0.1 V vs. SHE at pH 7.0) are lower by more than 0.2 V compared to the protein either in solution or immobilized with different anchoring strategies using uncharged SAMs. For the present electrostatic and covalent binding, this effect can, respectively, be ascribed to the negative charge of the SAM surfaces and to deletion of the positive charge of Lys/Arg residues due to amide bond formation which both selectively stabilize the more positively charged oxidized SLAC. Observation of enthalpy/entropy compensation within the series indicates that the immobilized proteins experience different reduction-induced solvent reorganization effects. The E°' values for the covalently attached SLAC are sensitive to three acid base equilibria, with apparent pK_a values of pK_a1ox = 5.1, pK_a1red = 7.5, pK_a2ox = 8.4, pK_a2red = 10.9, pK_a2ox = 8.9, pK_a2red = 11.3 possibly involving one residue close to the T1 center and two residues (Lys and/or Arg) along with moderate protein unfolding, respectively. Therefore, the E°' value of immobilized SLAC turns out to be particularly sensitive to the anchoring mode and medium conditions.

Keywords: electron transfer; protein voltammetry; protein-electrode linkage; redox thermodynamics; self-assembled monolayers; small laccase.

Figure 1

Cyclic voltammograms for wt Streptomyces coelicolor small laccase (SLAC) electrostatically bound on a polycrystalline gold electrode coated with a SAM of MUA (A), 1:1 MUA/MU (B), 1:2 MUA/MU (C), and 1:3 MUA/MU (D). CVs were recorded in 5 mM Tris-HCl buffer, 5 mM sodium perchlorate, pH 7. Scan rate: 0.05 V s⁻¹, T = 20 °C.

Figure 2

Cyclic voltammograms for wt Streptomyces coelicolor small laccase (SLAC) covalently bound to a SAM of MUA (A), 1:1 MUA/MU (B), 1:2 MUA/MU (C), and 1:3 MUA/MU (D) through NHS/EDC chemistry. CVs were recorded in 5 mM Tris-HCl buffer, 5 mM sodium perchlorate, pH 7. Scan rate: 0.05 V s⁻¹, T = 20 °C.

Figure 3

(A) Surface electrostatic potential of the homo-dimeric Streptomyces coelicolor small laccase (SLAC) showing neutral (white), positively (blue) and negatively (red) charged areas. The three monomers are labeled A, B, and C. The figure was prepared using the NOC 3.01 package from the X-ray structure (PDB code 3CG8) [42]. (B) Schematic representation of SLAC with a decreased opacity. The T1 copper ions are depicted as green dots. The Arg170 and Lys204 residues for all subunits are highlighted. The distances form the T1 copper center to the selected residues of subunit B are calculated from the crystallographic structure.

Figure 4

Cyclic voltammograms for SLAC covalently immobilized on MUA/MU SAM at different exposure times of the electrochemical cell (initially under argon) to air at normal atmospheric pressure. CVs were recorded in 5 mM phosphate buffer, 5 mM sodium perchlorate at pH 4.5 (A), 7.0 (B), and 9.6 (C). Scan rate: 0.05 V s⁻¹, T = 20 °C.

Figure 5

E°′ vs. T plots of Streptomyces coelicolor small laccase (SLAC) immobilized on a gold electrode coated with electrostatically (A) MUA (●), 3:1 MUA/MU (×), 1:1 MUA/MU (▲), 1:2 MUA/MU (□), or covalently (B) MUA(EDC/NHS) (●), 3:1 MUA/MU (EDC/NHS) (×), 1:1 MUA/MU (EDC/NHS) (▲), 1:2 MUA/MU (EDC/NHS) (□), respectively. pH = 7.0. Reduction entropy (ΔS°′) is calculated from the slope of the linear regression lines. The error bars are not shown because they have the same dimension of the symbols.

Figure 6

Enthalpy–entropy compensation plots for the reduction thermodynamics of Streptomyces coelicolor small laccase (SLAC) electrostatically and covalently immobilized on a gold electrode coated with MUA, 3:1 MUA/MU, 1:1 MUA/MU, 1:2 MUA/MU, MUA(EDC/NHS), 1:1 MUA/MU(EDC/NHS) at pH 7.0 (●), and covalently immobilized with 1:1 MUA/MU(EDC/NHS) at pH 8.4 and 9.6 (▲). T=293 K. Error bars are reported when available or have the same dimensions of symbols.

Figure 7

pH dependence of E°′ for Streptomyces coelicolor small laccase (SLAC) covalently bound to 1:1 MUA/MU gold electrode through EDC/NHS linkage procedure. 5 mM Tris-HCl buffer plus 5 mM sodium perchlorate, T = 20 °C. The error bars are not shown because they have the same dimension of the symbols.

Figure 8

ln k_s vs. 1/T plots of Streptomyces coelicolor small laccase (SLAC) covalently bound to 1:1 MUA/MU gold electrode through EDC/NHS linkage procedure at different pH values: pH = 7.0 (○), pH = 8.4 (●), pH = 9.6 (▲), and electrostatically immobilized on 1:1 MUA/MU gold electrode at pH = 7.0 (×). Activation enthalpies (ΔH^#) are calculated from the slope of the linear regression curves.

Scheme 1

Schematic representation of the different strategies that were employed for SLAC immobilization. Upper panel: electrostatic interaction. Lower panel: covalent immobilization via formation of amide bond.