Ga3+ Replacing of Fe3+ as a “Trojan Horse” Antimicrobial Strategy: Modeling the Process within Bacterial Catalase–Peroxidase KatG

Abstract Replacement of Fe3+ by Ga3+ has emerged as a promising “Trojan horse” antimicrobial strategy in response to the growing threat of antibiotic-resistant bacteria. Because many bacterial proteins hijack host iron to sustain essential redox processes, substitution with redox inactive Ga3+, which closely resembles Fe3+ in ionic radius but possesses only one oxidation state, can disrupt their activity. Among potential targets, bacterial catalase-peroxidase shows particular sensitivity to Fe3+/Ga3+ exchange. However, the structural determinants and thermodynamic driving forces governing this substitution remain poorly understood. Here we combine ad hoc force field development, molecular dynamics simulations, alchemical free energy calculations, and QM/MM methods to elucidate the molecular basis of Fe3+/Ga3+ exchange in the bacterial catalase-peroxidase KatG. Our simulations show that the axial coordination environment of the heme B cofactor is largely preserved upon metal substitution, while the distal ligand dynamically alternates between histidine and a coordinated water molecule. Energy decomposition analysis reveals residue specific differences in electrostatic and van der Waals interactions following metal exchange. Free energy calculations indicate that substitution of iron by gallium in the KatG active site is thermodynamically favorable. The presence of a coordinated water molecule emerges as a key structural feature in both metalated states, while distinct binding patterns contribute differently to the overall thermodynamic driving force. These results provide an atomistic description of gallium mediated metal substitution in KatG and offer mechanistic insight into gallium-based antimicrobial strategies, supporting the rational design of new iron-mimetic antibacterial agents.