Modeling the pre-oxidation stage of firefly bioluminescence mechanism

Here we present the first stage in the firefly luciferase (FLuc) chemiluminescence pathway, namely the mechanism of the reaction of luciferin with ATP and subsequent deprotonation of the formed luciferyl adenylate. For this purpose, we manually reconstructed the structure of the luciferin-ATP-Mg2+ complex from the luciferin-AMP-Mg2+ structure (PDB ID: 6K4D) and then performed structure refinement using classical molecular dynamics (MD) and quantum mechanics/molecular mechanics (QM/MM) methods. Based on our simulation we depict the roles of amino acids in the active site involved in the formation of the enzyme-substrate complex and the pre-oxidation steps of bioluminescence reaction. By QM/MM approach, we show that the adenylation reaction proceeds via an associative mechanism and leads to luciferyl adenylate, which is easily deprotonated and releases a proton either to His244 or to the pyrophosphate PPi formed during adenylation. Thus, we provide evidence that in the luciferin-AMP-Mg2+ complex, the luciferyl adenylate is already in a deprotonated state, which is confirmed by the crystal structures of this complex.