Improvement of the SOD activity of the Cu2+ complexes by hybridization with lysozyme and its hydrogen bond effect on the activity enhancement

Abstract

We prepared L-amino acids (L-valine and L-serine, respectively) based on the Schiff base Cu²⁺ complexes CuSV and CuSS in the absence/presence of hydroxyl groups and their imidazole-bound compounds CuSV-Imi and CuSS-Imi to reveal the effects of hydroxyl groups on SOD activity. The structural and spectroscopic features of the Cu²⁺ complexes were evaluated using X-ray crystallography, UV-vis spectroscopy, and EPR spectroscopy. The spectroscopic behavior upon addition of lysozyme indicated that both CuSV and CuSS were coordinated by the imidazole group of His15 in lysozyme at their equatorial position, leading to the formation of hybrid proteins with lysozyme. CuSS-Imi showed a higher SOD activity than CuSV-Imi, indicating that the hydroxyl group of CuSS-Imi played an important role in the disproportionation of O₂ ^- ion. Hybridization of the Cu²⁺ complexes CuSV and CuSS with lysozyme resulted in higher SOD activity than that of CuSV-Imi and CuSS-Imi. The improvements in SOD activity suggest that there are cooperative effects between Cu²⁺ complexes and lysozyme.

Keywords: Cu2+ complex; SOD activity; hybrid protein; hydrogen bond; lysozyme.

FIGURE 1

The Cu²⁺ center structure of the hybrid protein composed of the Cu²⁺ complex (CuST) and lysozyme.

FIGURE 2

Schematic structure of (A) CuSV and (B) CuSS prepared in this study.

FIGURE 3

ORTEP drawings of Cu²⁺ complex CuSV with a (A) 4-coordinated and a (B) 5-coordinated structure. The thermal ellipsoids are drawn at 50% probability. Hydrogen atoms are omitted for clarity. Selected bond length (Å) and bond angles (degree): Cu1−N1 = 1.9205 (16), Cu1−O1 = 1.8955 (15), Cu1−O2 = 1.9339 (16), Cu1−O3 = 1.9629 (15), N1−Cu1−O1 = 94.40 (7), O1−Cu1−O2 = 94.04 (7), O2−Cu1−O3 = 88.01 (7), N1−Cu1−O3 = 82.90 (7), N1−Cu1−O2 = 170.16 (7), O1−Cu1−O3 = 171.32 (7), for structure (A), Cu2−N2 = 1.9243 (17), Cu2−O5 = 1.9432 (14), Cu2−O6 = 1.9411 (15), Cu2−O7 = 1.9956 (14), Cu2−O9 = 2.3663 (16), N2−Cu2−O5 = 92.86 (6), O5−Cu2−O6 = 89.41 (7), O6−Cu2−O7 = 95.00 (7), N2−Cu2−O7 = 82.74 (6), N2−Cu2−O9 = 93.05 (7), O5−Cu2−O9 = 97.98 (6), O6−Cu2−O9 = 86.56 (7), O7−Cu2−O9 = 82.34 (6), N2−Cu2−O6 = 177.73 (7), O5−Cu2−O7 = 175.60 (6), for structure (B), respectively.

FIGURE 4

ORTEP drawings of Cu²⁺ complex CuSV-Imi with thermal ellipsoids drawn at 50% probability. Hydrogen atoms are omitted for clarity. Selected bond length (Å) and bond angles (degree): Cu−N1 = 1.927 (3), Cu−O1 = 1.909 (3), Cu−N2 = 1.961 (3), Cu−O2 = 1.971 (3), N1−Cu−O1 = 94.33 (12), O1−Cu−N2 = 91.54 (12), N2−Cu−O2 = 90.53 (11), N1−Cu−O2 = 82.91 (11), N1−Cu−N2 = 172.24 (13), O1−Cu−O2 = 171.11 (11).

FIGURE 5

UV-vis spectra in methanol solution (0.02 mM) of (A) CuSV (solid line), CuSV-Imi (dashed line) and (B) CuSS (solid line), CuSS-Imi (dashed line). Insert: Expanded UV-vis spectra in 500–800 nm region (2 mM).

FIGURE 6

Comparisons of measured (solid lines) and simulated (dashed lines) EPR spectra of (A) CuSV (top), CuSV-Imi (bottom) and (B) CuSS (top), CuSS-Imi (bottom). All samples were prepared as 1 mM solutions in 0.1 M phosphate buffer (pH: 7.0) in quarts tubes and measured at 77 K.

FIGURE 7

Emission spectra (λ_ex = 260 nm) of lysozyme (4 μM) in presence of various concentrations of (A) CuSV (0–4 μM), (B) CuSS (0–4 μM), and their Stern–Volmer plots [(C,D), respectively]. These I₀/I values were plotted by using their emission maxima (λ_em = 346 nm). All samples were prepared as 0.1 M phosphate buffer solution (pH = 7.0) and measured at 298 K.

FIGURE 8

UV-vis spectra due to d-d transitions of (A) CuSV (0.8 mM) and (B) CuSS (0.8 mM) in presence of various concentrations of lysozyme (0–0.8 mM). All samples were prepared as 0.1 M phosphate buffer solution (pH = 7.0) and measured at 298 K.

FIGURE 9

EPR spectra of (A) CuSV (top), measured and simulated 1:1 mixture of CuSV and lysozyme (middle, bottom), (B) CuSS (top), measured and simulated 1:1 mixture of CuSS and lysozyme (middle, bottom), and differential EPR spectra of (C) CuSV (top), CuSV-Imi (middle), 1:1 mixture of CuSV and lysozyme (bottom), (D) CuSS (top), CuSS-Imi (middle), 1:1 mixture of CuSS and lysozyme (bottom). All samples were prepared as 0.8 mM solution in 0.1 M phosphate buffer solution (pH = 7.0) in quarts tubes and measured at 77 K.

FIGURE 10

Cyclic voltammograms of (A) CuSV-Imi (solid line), CuSV@lysozyme (dashed line) and (B) CuSS-Imi (solid line), CuSS@lysozyme (dashed line). All samples were prepared as 1.0 mM solution in 0.1 M phosphate buffer solution (pH = 7.0). Glassy carbon, Pt wire, and Ag/AgCl electrodes were used as the working, counter, and reference electrodes, respectively and measured with a sweep rate of 100 mV/s. Potentials were converted from Ag/AgCl to NHE.

FIGURE 11

Theoretically proposed structures of (A) O₂ ⁻-bound CuSV-Imi and (B) O₂ ⁻-bound CuSS-Imi. Selected bond length (Å) and bond angles (degree): Cu−N1 = 2.022, Cu−O1 = 2.038, Cu−N2 = 2.397, Cu−O2 = 2.048, Cu−O4 = 1.975, N1−Cu−O1 = 90.156, O1−Cu−O4 = 92.369, O4−Cu−O2 = 96.667, O2−Cu−N1 = 81.359, N1−Cu−N2 = 106.462, O1−Cu−N2 = 90.077, O4−Cu−N2 = 88.433, O2−Cu−N2 = 89.232, N1−Cu−O4 = 164.894, O1−Cu−O2 = 170.915, for structure (A), and Cu−N1 = 1.940, Cu−O1 = 2.114, Cu−N2 = 1.993, Cu−O2 = 2.113, Cu−O5 = 2.169, N1−Cu−O1 = 91.113, O1−Cu−N2 = 93.168, N2−Cu−O2 = 92.353, O2−Cu−N1 = 80.806, N1−Cu−O5 = 98.791, O1−Cu−O5 = 95.293, N2−Cu−O5 = 86.225, O2−Cu−O5 = 128.491, N1−Cu−N2 = 173.086, O1−Cu−O2 = 136.136, for structure (B), respectively.

FIGURE 12

Estimated structure of O₂ ⁻-bound CuSS@lysozyme.