Theoretical Insights into the Reduction of Azurin Metal Site with Unnatural Amino Acid Substitutions

Copper-containing proteins play crucial roles in biological systems. Azurin is a copper-containing protein which has a Type 1 copper site that facilitates electron transfer in the cytochrome chain. Previous research has highlighted the significant impact of mutations in the axial Met121 of the copper site on the reduction potential. However, the mechanism of this regulation has not been fully established. In this study, we employed theoretical modeling to investigate the reduction of the Type 1 copper site, focusing on how unnatural amino acid substitutions at Met121 influence its behavior. Our findings demonstrated a strong linear correlation between electrostatic interactions and the reduction potential of the copper site, which indicates that the perturbation of the reduction potential is primarily influenced by electrostatic interactions between the metal ion and the ligating atom. Furthermore, we found that CF/π and CF…H interactions could induce subtle changes in geometry and hence impact the electronic properties of the systems under study. Interestingly, our calculations suggest the reduction potential of the 6-coordinated copper sites would be significantly more sensitive to the distance between the ion and equatorial ligands, compared to the 5-coordinated ones. Overall, this study offers valuable insights into the structural and electronic properties of the Type 1 copper site, which could potentially guide the design of future artificial catalysts.