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ELECTROCHEMICAL LUMINESCENCE WITH N(5)‐ETHYL‐4a‐HYDROXY‐3‐METHYL‐4a, 5‐DIHYDROLUMIFLAVIN. THE MECHANISM OF BACTERIAL LUCIFERASE
Author(s) -
Kaaret Thomas W.,
Bruice Thomas C.
Publication year - 1990
Publication title -
photochemistry and photobiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.818
H-Index - 131
eISSN - 1751-1097
pISSN - 0031-8655
DOI - 10.1111/j.1751-1097.1990.tb01976.x
Subject(s) - acetonitrile , chemiluminescence , quantum yield , electrochemistry , luminescence , chemistry , fluorescence , photochemistry , excited state , dimethylformamide , ring (chemistry) , electrode , electrochemiluminescence , redox , electrochemical cell , analytical chemistry (journal) , nuclear chemistry , inorganic chemistry , materials science , organic chemistry , optics , optoelectronics , physics , solvent , nuclear physics
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 le − reduction of the radical 4a‐FlHOH t . 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‐dihydroluminfiavin (4a‐FlEtOH) have been determined in dry N,N‐dimethylformamide (DMF). Photon counting was carried out during the le − 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 le oxidation of 4a‐FlEtOH + → 4a‐FlEtOH + and le − 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 le − reductive capture of 4a‐FlEtOrP as lO −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 − 3 × 10 −5 . No electrogenerated chemiluminescence is observed on the electrochemical recycling of FlEt + → FlEt 2+ and FlEt 2+ → FlEt + .