SPECTRAL DETECTION OF AN INTERMEDIATE PRECEDING THE EXCITED-STATE IN THE BACTERIAL LUCIFERASE REACTION

The bioluminescent reaction of luciferase isolated from Vibrio harveyi , strain M17, was initiated by mixing the luciferase-bound flavin 4a-h ydroperoxide intermediate, purified in advance, with a long-chain alde hyde (dodecanal or octanal) at -4-degrees-C. Measurements of absorbanc e changes from 300 to 600 nm during the course of the reaction reveale d the existence of three sets of isosbestic points and three kinetic p hases, the second of which parallels kinetically the decay of biolumin escence, measured concurrently. The absorbance changes in this second step and the decay of light emission exhibited similar deuterium isoto pe effects; this is postulated to be the step giving rise to the excit ed state and the enzyme-bound flavin 4a-hydroxide. The first step of t he reaction, however, did not show an isotope effect; the intermediate thereby formed, observed here for the first time, is postulated to co rrespond to the luciferase-bound flavin 4a-peroxyhemiacetal.