Computations Reveal How Firefly Luciferase Governs Side Reactivity of Superoxide

The dual nature of superoxide (O₂•⁻) as both a damaging oxidant and a common intermediate in oxygen-dependent enzymatic reactions raises a fundamental question about how enzymes control its reactivity. Firefly luciferase (FLuc), which productively utilizes O₂•⁻ to drive its light-emitting reaction, provides an excellent model system to elucidate these control mechanisms. In this work, using a combination of quantum chemical, QM/MM, and QM/MM MD simulations, we demonstrate that this selectivity is governed by a preorganized active-site architecture. We show that a hydrogen-bonding network, primarily with the F246 backbone and active-site water molecules, stabilizes O₂•⁻. At the same time, the G245-F246 backbone and F246 side chain sterically block unproductive additions of O₂•⁻ to the deprotonated luciferyl adenylate radical and orient O₂•⁻ for selective formation of the dioxetanone─the key intermediate of the light-emitting pathway. We also identify how the H244 side chain, protonated in the preceding reaction steps, can trigger a major unproductive side reaction producing the hydroperoxyl radical and subsequently hydrogen peroxide, and propose that this pathway may be potentially regulated by the pyrophosphate cofactor acting as a scavenger for excessive protons in the active site. Finally, we establish that the Mg²⁺ ion is essential for the final dioxetanone ring closure by charge neutralization, which reduces the electrostatic repulsion in the AMP cleavage step and prevents the formation of an adenylated dioxetanone analogue unproductive in light emission.