QM/MM Molecular Modeling Reveals Mechanism Insights into Flavin Peroxide Formation in Bacterial Luciferase

Bacterial luciferase (Lux) catalyzes oxidation of reduced flavin mononucleotide (FMN) and aldehyde to form oxidized FMN and carboxylic acid via molecular oxygen with concomitant light generation. The enzyme is useful for various detection applications in biomedical experiments. Upon reacting with oxygen, the reduced FMN generates C4a-peroxy-FMN (FMNH-C4a–OO^–) as a reactive intermediate, which is required for light generation. However, the mechanism and control of FMNH-C4a–OO^– formation are not clear. This work investigated the reaction of FMNH-C4a–OO^– formation in Lux using QM/MM methods. The B3LYP/6-31G*/CHARMM27 calculations indicate that Lux controls the formation of FMNH-C4a–OO^– via the conserved His44 residue. The steps in intermediate formation are found to be as follows: (i) H⁺ reacts with O₂ to generate ⁺OOH. (ii) ⁺OOH attacks C4a of FMNH^– to generate FMNH-C4a–OOH. (iii) H⁺ is transferred from FMNH-C4a–OOH to His44 to generate FMNH-C4a–OO^– while His44 stabilizes FMNH-C4a–OO^– by forming a hydrogen bond to an oxygen atom. This controlling key mechanism for driving the change from FMNH-C4a–OOH to the FMNH-C4a–OO^– adduct is confirmed because FMNH-C4a–OO^– is more stable than FMNH-C4a–OOH in the luciferase active site.