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

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 (E(obs)) 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 (E(calc)). 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. A good correlation was also found between E(obs) and the literature values of the 2-electron oxidation/reduction potentials (E(redox)) of the couple FI(ox)/1,5-dihydro-FI(red) for the flavin derivatives having the same substituent at position 8. The effects of the substituent in position 8 on the redox properties of 1,5-dihydro- and 4a,5-dihydroflavins are thus essentially the same. This indicates that the earlier use of readily available redox potentials for FI(ox)/1,5-dihydro-FI(red) for studying reactions involving the 4a,5-dihydro isomer was sound.