title: Mechanism of bacterial bioluminescence: 4a,5-dihydroflavin analogs as models for luciferase hydroperoxide intermediates and the effect of substituents at the 8-position of flavin on luciferase kinetics.
abstract: Bioluminescence catalyzed by bacterial luciferases was measured using FMN, iso-FMN (6-methyl-8-nor-FMN), and FMN analogs carrying the following substituents at position 8: -H, -Cl, -F, SMe, SOMe, -SO2Me, or -OMe. The first-order rate constants for the decay of light emission correlate with the one-electron oxidation potentials of the 4a,5-dihydro forms of the FMN analogs. To determine the values of these potentials, isoalloxazine (flavin) derivatives having the 4a,5-propano-4a,5-dihydro structure and -H, -CH3, -Cl, -OCH3, and -NH2 as substituents at position 8 have been synthesized as models for the 4a-peroxy-4a,5-dihydroflavin intermediates occurring during catalysis by the flavin-dependent monooxygenase luciferase. The tetrahydropyrrole ring between positions 4a and 5 of these isoalloxazine derivatives stabilizes the 4a,5-dihydroflavin by impeding formation of the thermodynamically more stable 1,5-dihydro form. One-electron oxidation potentials (Eobs) were measured by cyclic voltammetry and used to determine the empirical coefficients in the Swain equation. On the basis of this, the one-electron oxidation potentials of 4a,5-propano-4a,5-dihydro analogs with other substituents in position 8 were calculated (Ecalc). The bioluminescence reaction rate is fastest with FMN analogs of lowest oxidation potential; i.e., the slope of the correlation is negative. This indicates that in the rate-limiting step the 4a,5-dihydroflavin moiety donates negative charge. The results are compatible with an intramolecular, chemically initiated electron exchange luminescence mechanism for the bacterial luciferase reaction.(ABSTRACT TRUNCATED AT 250 WORDS)
authors.affiliation: Department of Biology, University of Konstanz, Germany.
authors.forename: J W
authors.initials: JW
authors.lastname: Eckstein
authors.forename: J W
authors.initials: JW
authors.lastname: Hastings
authors.forename: S
authors.initials: S
authors.lastname: Ghisla
mesh_terms: D004563:Electrochemistry; D005486:Flavin Mononucleotide / Q000737:chemistry*; D005415:Flavins / Q000737:chemistry*; D007700:Kinetics; D008156:Luciferases / Q000737:chemistry*; D008163:Luminescent Measurements; D009682:Magnetic Resonance Spectroscopy; D010084:Oxidation-Reduction; D010776:Photobacterium / Q000378:metabolism; D013056:Spectrophotometry, Ultraviolet; D013816:Thermodynamics; D014733:Vibrio / Q000378:metabolism
chemical_list: D005415:Flavins; D005486:Flavin Mononucleotide; D008156:Luciferases