Protonation status and control mechanism of flavin–oxygen intermediates in the reaction of bacterial luciferase

Abstract

Bacterial luciferase proceeds bioluminescent reaction by generating a reactive intermediate of flavin C4a‐oxygen adduct. The intermediate is first generated in a protonated form of flavin C4a‐hydroperoxide, which is unable to react with an aldehyde. The active site His44 functions as an essential proton abstractor to convert the flavin C4a‐hydroperoxide to a bioluminescent active flavin C4a‐peroxide.

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

“…2 Although the αHis44Ala variant was able to form FMNH-C4a−OOH and its stability was not significantly affected, the intermediate generated by the αHis44Ala variant was inactive and could not proceed to generate bioluminescence because the pK a value of the terminal −OOH of FMNH-C4a−OOH is elevated. 2 However, the exact mechanistic role of His44 (e.g., whether it acts as a direct proton abstractor or how it stabilizes FMNH-C4a−OO − or lowers the −OOH pK a value) is not clear. The protonation state of the terminal peroxy group in flavin C4a−OOH found in flavin-dependent monooxygenases is important for controlling the function of this flavin-oxygen adduct, to be an electrophile or a nucleophile.…”

“…During the reaction catalyzed by Lux, light (λ max 490 nm) is generated concomitantly. 1,2 Lux is a heterodimeric enzyme, composed of αand β-subunits. The active site is located in the α-subunit, while the β-subunit is required to maintain active conformation.…”

“…The deprotonated form of flavin C4a-peroxide (FMNH-C4a−OO − ) acts as a nucleophile to attack an aldehyde to produce flavin C4a-peroxyhemiacetal (FMNH-C4a−OOR) prior to the O−O cleavage to yield carboxylic acid and an active luminescent species of excited state C4ahydroxyflavin (FMNH-C4a−OH*). 2 Light is liberated when the FMNH-C4a−OH* transforms to the FMNH-C4a−OH ground state. The FMNH-C4a−OH then decays to form FMN and water as final products (Figure 1).…”

“…However, this suggestion contradicts recent experimental findings which indicate that αHis44 may accept H + from the first generated intermediate FMNH-C4a−OOH to form the active FMNH-C4a−OO − adduct. 2 The FMNH-C4a−OO dramatic reduction of light yield. 1 Change of αCys106, which is close to the C4a position on the re-face of the isoalloxazine ring to Val resulted in a 50-fold decrease in light output compared to the wild-type enzyme.…”

A Flavin-Dependent Monooxygenase Mediates Divergent Oxidation of Rifamycin

“…His44 is a catalytic residue in the intensively studied bacterial luciferase LuxA and varies in the inactive partner LuxB (Figure S18B). , Homologous modeling from 3,6-diketone camphane monooxygenase (20% identical to Rif-Orf17) yielded a structure of Rif-Orf17 (100% confidence) displaying conserved TIM-barrel folding with the FMN cofactor (Figure S19). In addition to H44 and H45, several histidines were noticed around the si -face of the isoalloxazine (Figure S19F), which are absent in other LLMs (Figure S18B) and were supposed to be involved in the multiple functions of Rif-Orf17.…”