Spectral properties of an oxygenated luciferase-flavin intermediate isolated by low-temperature chromatography.

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.