Mechanism of Bacterial Bioluminescence: 4a,S-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 (6methyl-8-nor-FMN), and FMN analogs carrying the following substituents a t position 8: -H, -C1, -F, S M e , S O M e , S 0 2 M e , or -0Me. 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 thevalues of these potentials, isoalloxazine (flavin) derivatives having the 4a,5-propano-4a,5-dihydro structure and -H, -CH3, -C1, -OCH3, and -NH2 as substituents a t 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 4aJ-dihydroflavin by impeding formation of the thermodynamically more stable 1,5-dihydro form. One-electron oxidation potentials 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; Le., 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 EOb and the literature values of the 2-electron oxidation/reduction potentials (Erdox) of the couple FIox/1,5-dihydro-FIr~ for the flavin derivatives having the same substituent a t position 8. The effects of the substituent in position 8 on the redox properties of 1,5-dihydroand 4a,5-dihydroflavins are thus essentially the same. This indicates that the earlier use of readily available redox potentials for FI,/ 1,5-dihydro-FIrd for studying reactions involving the 4aS-dihydro isomer was sound. Bacterial luciferase (E.C. 1.14.14.3) catalyzes the monooxygenation of long-chain aldehyde to the corresponding carboxylic acid concomitant with production of light; the cosubstrate is FMNH2,’ which is oxidized to FMN in the course of the following reaction (Hastings et al., 1985): FMNH, + RCHO + 0, FMN + RCOOH + luciferase