Quercetin-loaded ZnO nanoparticles modulate the pH-dependent structural features, conformational stability, and peroxidase activity of horse ferricytochrome c.

Quercetin-loaded zinc oxide nanoparticles (Q/ZnO NPs) were synthesized and characterized using SEM, FTIR, XRD, DLS Zeta Potential, and UV-visible spectroscopy. This study investigates the impact of Q/ZnO NPs on the pH-dependent local, structural, and global stability of horse ferricytochrome c (FeIII-Cytchm c). Analysis of the effects of 0.05 mg/mL Q/ZnO NPs on the spectroscopic features, alkaline pH-titrations, and thermal/chemical-denaturation profiles of FeIII-Cytchm c yielded significant insights: (i) Q/ZnO NPs do not affect the FeIII-Met80 bond interactions and protein structure at pH 7 and 10.5, but they reinforce the base-induced structural destabilization of the protein at pH ~ 12 (ii) these NPs do not influence the alkaline transition of FeIII-Cytchm c but promote the pH-induced structural-unfolding (iii) Q/ZnO NPs decrease the local (Met80-Fe3+ link) and structural stability at pH 7, and global and structural stability at pH 10.5 and 12. Molecular docking studies suggest that Q/ZnO NPs form non-covalent (H-bond and π-bond) binding interactions with FeIII-Cytchm c. Additionally, ITC and Trp-fluorescence (Stern-Volmer plot) studies of FeIII-Cytchm c with Q/ZnO NPs at pH 7, 10.5, and 12 indicated that these NPs form binding interactions with the protein at alkaline pH. Furthermore, Q/ZnO NPs do not alter the peroxidase activity of FeIII-Cytchm c. Analysis of the effects of Q/ZnO NPs on the Trp-fluorescence lifetime kinetics of FeIII-Cytchm c suggested that these NPs shorten the average fluorescence lifetime of the protein at pH 7.0, 10.5, and 12, indicating that dynamic interactions of these NPs with the protein reduce the local, structural, and global stability of the protein.