Structure and catalytic inactivity of the bacterial luciferase neutral flavin radical

A luciferase-bound neutral flavin semiquinone radical can be formed upon the oxidation of the luciferase-FMNH2 complex by molecular oxygen. This species can also be formed anaerobically by comproportionation of FMN and FMNH2 in the presence of luciferase. The radical is kinetically stable (t1/2 approximately 20 h at 0 degree C in air; the Arrhenius delta H not equal to decay being about 170 kJ/mol) and can be prepared in pure form by Sephadex G-25 chromatography at 0-4 degrees C. The pure enzyme-bound radical is inactive for light emission either with or without aldehyde, and is not in (relevantly rapid) equilibrium with the luciferase 4a-peroxyflavin, the active intermediate in the bioluminescent reaction.