Bacterial bioluminescence: equilibrium association measurements, quantum yields, reaction kinetics, and overall reaction scheme.
The characteristics of the bioluminescence reactions with bacterial luciferase from two different cell types, Photobacterium fischeri and Beneckea harveyi, are reported. The reduced flavine mononucleotide (FMNH2)-luciferase association constant, directly measured by equilibrium dialysis and gel filtration is the same for both luciferases, 3 times 10(-4) Mminus1 at romm temperature, and is significantly different from the kinetic reciprocal Michaelis-Menten constant. The luciferase bioluminescence quantum yield for the highest activity preparations is the same as for the aldehyde. Rapid stopped-flow observations show that the oxidation of FMNH2 in the presence of sufficient luciferase to outcompete autoxidation, is bimodal. A long-lived intermediate, formed before reaction with aldehyde, has an activation energy for decay of 35 kcal mol-1, much greater than for the light reaction, 14 kcal mol-1. The ratio of bioluminescence quantum yields with respect to aldehyde and FMNH2 is independent of temperature, however, and also of aldehyde chain lenght longer than octanal, pH (6.5-8), and type of luciferase and its specific activity. Even when the aldehyde concentration limits the rate of the light reaction, the quantum yield of the long-lived intermediate is unchanged, and together these data mean that, under the optimal conditions chosen for quantum yield measurements, no dark side reactions effectively compete with the main reaction leading to light emission. A series of reactions involving one-electron steps and the sequential oxidation of two FMNH2 molecules is postulated for the formation of the long-lived intermediate.
D000447:Aldehydes; D001665:Binding Sites; D002850:Chromatography, Gel; D003956:Dialysis; D005486:Flavin Mononucleotide; D006088:Gram-Negative Aerobic Bacteria / Q000201:enzymology*; D007700:Kinetics; D008027:Light; D008163:Luminescent Measurements; D010084:Oxidation-Reduction; D010776:Photobacterium / Q000201:enzymology*; D011485:Protein Binding; D011789:Quantum Theory; D013696:Temperature
D000447:Aldehydes; D005486:Flavin Mononucleotide