Influence of Ligand Complexity on the Spectroscopic Properties of Type 1 Copper Sites: A Theoretical Study

Multi-copper oxidases (MCOs) are enzymes of significant interest in biotechnology due to their efficient catalysis of oxygen reduction to water, making them valuable in sustainable energy production and bio-electrochemical applications. This study employs time-dependent density functional theory (TDDFT) to investigate the electronic structure and spectroscopic properties of the Type 1 (T1) copper site in Azurin, which serves as a model for similar sites in MCOs. Four model complexes of varying complexity were derived from the T1 site, including 3 three-coordinate models and 1 four-coordinate model with axial methionine ligation, to explore the impact of molecular branches and axial coordination. Calculations using ωB97X-D3 functional, def2-TZVP basis set, and conductor-like polarizable continuum model (CPCM) solvation model reproduced key experimental spectral features, with increased model complexity improving agreement, particularly for the ~400 cm^-1 band splitting in resonance Raman spectra. This work enhances our understanding of T1 copper sites' electronic properties and spectra, bridging the gap between simplified models and complex proteins. The findings contribute to the interpretation of spectroscopic data in blue copper proteins and may inform future studies on similar biological systems.