DFT investigation of metal coordination and reactivity in minimal metalloenzyme models

Metalloenzymes achieve catalytic functionality by precisely controlling their metal coordination environments through structural constraints. However, the influence of structural rigidity on metal substitution and its impact on enzyme structure and reactivity has not been fully elucidated. To address this, we investigated how structural constraints affect metal coordination geometry, energetics, and reactivity within the active site of human carbonic anhydrase II (CA II) using DFT. We constructed semi-constrained models of metal substituted CA II from their X-ray crystal structures containing Zn²⁺ (native), Cu²⁺, Ni²⁺, and Co²⁺. Semi-constrained models were constructed to mimic the microenvironment of the protein active site, and multiple DFT methods were benchmarked to identify an accurate and efficient computational approach. Structural constraints lead to a rugged energy landscape with multiple local minima, and we found that upon metal substitutions, the competition between the structural constraints and the intrinsic coordination chemistry leads to diverse consequences in final geometry. We also found that the native metal ion (Zn²⁺) in metalloenzymes CA II does not always exhibit the strongest binding among the metal ions tested; instead, the trends follow the Irving-Williams series. However, electrophilicity analysis revealed that constrained geometries modulate the electronic reactivity of the metal center, with Zn²⁺ consistently exhibiting the highest electrophilicity, and this explains the evolutionary optimization of the metalloenzyme. These findings enhance our understanding of metal coordination under structural constraints and provide a computational basis for exploring metal substitutions in artificial metalloenzymes.