title: Electrochemical luminescence with N(5)-ethyl-4a-hydroxy-3-methyl-4a, 5-dihydrolumiflavin. The mechanism of bacterial luciferase.
abstract: It has been proposed in the literature that the chemiluminescence of the flavoenzyme of bacterial luciferase is caused by a chemically initiated electron-exchange luminescence mechanism which provides an excited 4a-hydroxy-4a,5-dihydroflavin ([4a-FlHOH]*) as product of 1e- reduction of the radical 4a-FlHOH+.. Electrochemical/photon counting experiments were performed to assess the feasibility of this proposal. Potentials for step-wise oxidation of N(5)-ethyl-4a-hydroxy-4a,5-dihydroluminflavin (4a-FlEtOH) have been determined in dry N,N-dimethylformamide (DMF). Photon counting was carried out during the 1e- reduction of 4a-FlEtOH+.in both DMF and acetonitrile by use of an apparatus consisting of a photocell mounted below a Pt ring-disk electrode. By use of the ring-disk electrode a steady state concentration of [4a-FlEtOH]* could be maintained by continuous 1e- oxidation of 4a-FlEtOH----4a-FlEtOH+.and 1e- reduction of 4a-FlEtOH+.----4a-FlEtOH. A maximum of 14% collection (theoretical maximum is 18%) of FlEtOH.+ at the ring electrode was obtained below 5000 rotations per minute. Calibration of the apparatus using 9,10-diphenylanthracene allowed approximation of the quantum yield for 1e- reductive capture of 4a-FlEtOH+.as 10(-6) to 10(-4) in DMF and 10(-7) to 10(-5) in acetonitrile. No fluorescence for 4a-FlEtOH in DMF could be observed; if fluorescent, the efficiency of 4a-FlEtOH can be no greater than approximately 3 x 10(-5). No electrogenerated chemiluminescence is observed on the electrochemical recycling of FlEt+----FlEt2+ and FlEt2+----FlEt+.
authors.affiliation: Department of Chemistry, University of California, Santa Barbara 93106.
authors.forename: T W
authors.initials: TW
authors.lastname: Kaaret
authors.forename: T C
authors.initials: TC
authors.lastname: Bruice
mesh_terms: D004563:Electrochemistry; D005415:Flavins / Q000378:metabolism*; D008156:Luciferases / Q000378:metabolism*; D049449:Luminescence; D014733:Vibrio / Q000201:enzymology*
chemical_list: D005415:Flavins; C064924:N(5)-ethyl-4a-hydroxy-3-methyl-4a,5-dihydrolumiflavin; D008156:Luciferases