β‐Deuterium Isotope Effects on Firefly Luciferase Bioluminescence

A 5,5‐d(2)‐luciferin was prepared to measure isotope effects on reactions of two intermediates in firefly bioluminescence: emission by oxyluciferin and elimination of a putative luciferyl adenylate hydroperoxide to dehydroluciferin. A negligible isotope effect on bioluminescence provides further support for the belief that the emitting species is the keto‐phenolate of oxyluciferin and rules out its excited‐state tautomerization, one potential contribution to a bioluminescence quantum yield less than unity. A small isotope effect on dehydroluciferin formation supports a single‐electron‐transfer mechanism for reaction of the luciferyl adenylate enolate with oxygen to form the hydroperoxide or dehydroluciferin. Partitioning between the dioxetanone intermediate (en route to oxyluciferin) and dehydroluciferin is determined, not by the fate of the hydroperoxide, but by that of the radical formed from luciferyl adenylate, and the kinetic isotope effect (KIE) reflects H‐atom abstraction by superoxide.