Spectral Properties of an Oxygenated Luciferase—Flavin Intermediate Isolated by Low-Temperature Chromatography
- 1 December 1973
- journal article
- Published by Proceedings of the National Academy of Sciences in Proceedings of the National Academy of Sciences
- Vol. 70 (12), 3468-3472
- https://doi.org/10.1073/pnas.70.12.3468
Abstract
Bacterial luciferase catalyzes the oxidation of reduced flavin mononucleotide by molecular oxygen; long-chain aldehyde is required for light emission. At 20 degrees the bioluminescence has a lifetime of tens of seconds, while excess reduced flavin is removed by way of nonenzymatic autoxidation in less than a second. This observation indicates the existence of a long-lived enzyme intermediate, which has been postulated to be a peroxide of the enzyme-bound reduced flavin. This intermediate was isolated and studied at low temperature (-20 degrees ), where it has a lifetime measured in days. It has an absorption with a single band peaking at 372 nm, and fluorescence emission centered at about 485 nm, which might be expected for the postulated flavin peroxide. Upon conversion to product, flavin mononucleotide-like absorption and fluorescence appear, supporting the postulate that flavin turns over in the reaction. Upon injection into buffer at 20 degrees with added aldehyde, bioluminescence occurs. Based on a stoichiometry of one flavin per luciferase molecule, the specific activity of the intermediate is equal to that of pure luciferase.Keywords
This publication has 20 references indexed in Scilit:
- Ionic Strength and Protonic Activity of Supercooled Solutions Used in Experiments with Enzyme SystemsJournal of Biological Chemistry, 1973
- Enzymology at sub-zero temperaturesMolecular and Cellular Biochemistry, 1973
- Reactions Involved in Bioluminescence Systems of Limpet ( Latia neritoides ) and Luminous BacteriaProceedings of the National Academy of Sciences, 1972
- Binding site determination from kinetic data. Reduced flavin mononucleotide binding to bacterial luciferase.1971
- A stable, inexpensive, solid-state photomultiplier photometerAnalytical Biochemistry, 1971
- Reduktive Photoalkylierung des Flavinkerns und Flavinkatalysierte Photodecarboxylierung von Phenylacetat Studien in der Flavinreihe. 15. Mitteilung [1]Helvetica Chimica Acta, 1967
- ON THE MOLECULAR MECHANISM OF BIOLUMINESCENCE, I. THE ROLE OF LONG-CHAIN ALDEHYDEProceedings of the National Academy of Sciences, 1964
- Intermediates in the Bioluminescent Oxidation of Reduced Flavin MononucleotideJournal of Biological Chemistry, 1963
- Quantum efficiency determinations on components of the bacterial luminescence systemBiochimica et Biophysica Acta, 1957
- A new method for preparing flavin-adenine dinucleotideBiochemical Journal, 1953