Bacterial Bioluminescence: Spectral Study of the Emitters in the In Vivo Reaction

Transient fluorescent species are observed in the bioluminescent reactions of three reduced flavin mononucleotides with aliphatic aldehydes and oxygen, catalyzed by bacterial luciferase. In each case the fluorescence spectral distribution is similar to that of the bioluminescence but is readily distinguishable from it on the basis of a significantly greater signal strength. The corrected bioluminescence maxima using Beneckea harveyi luciferase are 479 nm (iso-FMNH2), 490 nm (FMNH2), and 560 nm (2-thio-FMNH2). In an ethanol glass at 77 K, 2-thioriboflavin is fluorescent (ϕF = 0.03, λmax = 562 nm). These results are interpreted by a sensitized chemiluminescence mechanism in which the flavins bound to luciferase act as acceptors of excitation energy. For 2-thio-FMNH2, this acceptor species appears to be the oxidized 2-thio-FMN on the basis of the spectral evidence, whereas for the other flavins, some form of reduced species is a more likely candidate