Multi-copper oxidases (MCOs) are enzymes of significant interest in sustainable energy production and bio-electrochemical applications due to their efficient catalysis of oxygen reduction. This talk presents a comprehensive computational investigation of MCO Type 1 (T1) copper sites, progressing from small molecule mimics to whole protein systems.
The initial study employed time-dependent density functional theory (TDDFT) with hybrid functionals to examine Cu²⁺ thiolate complexes mimicking T1 sites. Experimental electronic absorption and resonance Raman (RR) spectra were accurately reproduced, including the characteristic ~400 cm^-1 Cu-S stretching mode. Charge transfer (qCT) was analyzed in terms of the transition density matrix and natural transition orbitals (NTOs).
Following the mimic studies, calculations were extended to Azurin, a blue copper protein. Range-corrected functionals were employed to account for long-range effects. The T1 site was systematically truncated to create complexes of increasing complexity, allowing exploration of molecular branch impacts on spectral features. This systematic approach provided crucial insights for defining the QM region in subsequent QM/MM calculations.
Finally, quantum mechanics/molecular mechanics (QM/MM) calculations were performed on Fet3p, a full MCO system, continuing to use range-corrected functionals. The QM region included the T1 site and extended towards the trinuclear copper cluster (TNC) site. After molecular dynamics simulations using the CHARMM36 force field, normal mode and TDDFT calculations were conducted on the enzyme's active site. The QM/MM approach successfully reproduced the experimental three-peak pattern around 400 cm^-1 in Raman spectra and captured key features of absorption spectra. Experimental spectra of the small laccase (SLAC) protein were further characterized using Fet3p as a model, providing insights into structural factors influencing copper site properties across different MCOs.
This progression from small mimics to whole protein systems demonstrates the power of utilizing freely accessible computational tools to elucidate the electronic structure and spectroscopic properties of MCOs. These findings contribute to the interpretation of spectroscopic data in blue copper proteins and may inform future studies on similar biological systems, ultimately supporting the development of MCO-based sustainable technologies.

Carol Korzeniewski

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