Isolation and properties of bacterial luciferase-oxygenated flavin intermediate complexed with long-chain alcohols.

Nonsubstrate long-chain aliphatic alcohols, carboxylic acids, and their methyl esters were found to complex reversibly with and stabilize an oxygenated flavin-luciferase intermediate, with alcohols being more effective in stabilizing the intermediate. Dissociation constants for the binding of alcohols to luciferase intermediate are in the order of K8 greater than K10 greater than K12 congruent to K14 where the subscripts represent the numbers of carbon atoms of various alcohols. Thermodynamic activation parameters for the decay of oxygenated flavin-luciferase intermediate complexed with alcohols or aldehydes were determined, and similarities were noted between alcohol and aldehyde complexes. Luciferase intermediate complexes formed with 1-decanol and 1-tetradecanol were isolated at 0 degrees C in neutral phosphate buffer, and both showed absorption properties characteristic of 4a-substituted dihydroflavins. The 1-tetradecanol-intermediate species contained one favin per luciferase molecule. Initially this complex was weakly fluorescent, but upon exposure to 370-nm light it was transformed to a highly fluorescent species. The latter shows a fluorescence excitation peak at 370 nm, and its fluorescence emission (lambda max 505 nm) and quantum yield (0.17) closely correspond to that of bioluminescence in vitro. Both the weakly and the highly fluorescent species exhibit full bioluminescence activities when reacted with decanal.