Fast kinetics of photoprotein emitting species.

Coelenterazine is the most common substrate for light-emitting reactions identified in luminous marine organisms. Among bioluminescent proteins engaging coelenterazine as a luciferin, Ca-regulated photoproteins form stable enzyme-substrate complexes offering thereby a unique opportunity to study their bioluminescence reactions in detail. Here, we used stopped-flow kinetics to investigate the formation of the emitters of recombinant aequorin, obelin, and W92F obelin activated with coelenterazine, as well as aequorin activated with coelenterazine-e. Based on the presence of up to four different spectral components, a modified unanimous kinetic model describing the bioluminescence reaction of Ca-regulated photoproteins is presented. The neutral, amide anionic, and phenolate anionic excited states of coelenteramide are proposed to originate from different pathways of dioxetanone decomposition with competing rates of proton transfer, radiation, and population and consequently to act as independent emitters in photoprotein bioluminescence.