The transfer of reduced flavin mononucleotide from luxg oxidoreductase to luciferase occurs via free diffusion

Abstract

Bacterial luciferase (LuxAB) is a two-component flavin mononucleotide (FMN)-dependent monooxygenase that catalyzes the oxidation of reduced FMN (FMNH-) and a long-chain aliphatic aldehyde by molecular oxygen to generate oxidized FMN, the corresponding aliphatic carboxylic acid, and concomitant emission of light. The LuxAB reaction requires a flavin reductase to generate FMNH- to serve as a luciferin in its reaction. However, FMNH- is unstable and can react with oxygen to generate H 2O2, so that it is important to transfer it efficiently to LuxAB. Recently, LuxG has been identified as a NADH:FMN oxidoreductase that supplies FMNH- to luciferase in vivo. In this report, the mode of transfer of FMNH- between LuxG from Photobacterium leiognathi TH1 and LuxABs from both P. leiognathi TH1 and Vibrio campbellii (PlLuxAB and VcLuxAB, respectively) was investigated using single-mixing and double-mixing stopped-flow spectrophotometry. The oxygenase component of p- hydroxyphenylacetate hydroxylase (C2) from Acinetobacter baumannii, which has no structural similarity to LuxAB, was used to measure the kinetics of release of FMNH- from LuxG. With all FMNH- acceptors used (C 2, PlLuxAB, and VcLuxAB), the kinetics of FMN reduction on LuxG were the same, showing that LuxG releases FMNH- with a rate constant of 4.5-6 s-1. Our data showed that the kinetics of binding of FMNH -to PlLuxAB and VcLuxAB and the subsequent reactions with oxygen were the same with either free FMNH- or FMNH- generated in situ by LuxG. These results strongly suggest that no complexes between LuxG and the various species are necessary to transfer FMNH- to the acceptors. The kinetics of the overall reactions and the individual rate constants correlate well with a free diffusion model for the transfer of FMNH- from LuxG to either LuxAB. © 2013 American Chemical Society.