Revisiting Oxygen Transport Features of Hemocyaninwith NEVPT2 Level QM/MM Calculations

This study explores the oxygen-binding mechanism and the potential peroxo-to-bis-μ-oxo isomerization in hemocyanin (Hc) using a quantum mechanics/molecular mechanics (QM/MM) approach at the multireference NEVPT2 level of theory (QM[NEVPT2]/MM). Our results support the previously proposed mechanism for Hc oxygen binding, involving two nearly simultaneous electron-transfer (ET) steps and a triplet–singlet intersystem crossing (ISC). However, we find that the first ET step occurs prior to ISC, resulting in the formation of a stable singlet superoxide intermediate through a low-energy barrier. The second ET leads to the formation of a singlet oxy-hemocyanin species featuring the characteristic peroxo–Cu₂O₂ “butterfly” core. Moreover, QM[NEVPT2]/MM simulations reveal a lower-energy barrier for the peroxo-to-bis-μ-oxo isomerization compared with density functional theory (DFT), although the peroxo form remains energetically favored within the protein environment. These findings offer new insights into the behavior of the hemocyanin active site, highlighting the importance of considering both the electronic correlation and the protein environment in accurately modeling copper–oxygen interactions in biological systems.