Metal ion mediated inhibition of firefly bioluminescence: A possibility via a quaternary complex

D(-) Luciferin, interacts with different metal ions to produce colourless s oluble salts with absorption spectra broader, intense and red shifted as co mpared to those of the parent compound. The equilibrium constants for the l uciferin-metal ion system vary in the order, depository divalent transition metal ions > alkali metal ions. The equilibrium constants for the ternary complexes formed between metal ions and a mixture of luciferin and lucifera se are larger than that of binary complexes but vary in the same order. Ste ady state fluorometric titration's of luciferin further confirmed its compl exation with metal ions. The single absorption maximum of firefly luciferas e at 278 nm originating from tyrosine was split into a doubler in presence of transition metal ions. The absorption maximum at lower wavelength is att ributed to the H-bond raptured free tyrosine denatured conformation of the luciferase while the longer wavelength band to tyrosine-transition metal io n complex. Difference spectra of luciferase metal ion complex yielded chang e in the molar extinction coefficients from which the number of tyrosine mo lecules exposed to aqueous solution by the perturbant metal ions are evalua ted following the Donovan model. The number of tyrosine molecules exposed t o the aqueous medium as a result of conformational change in the enzyme are 4, 3, 3, 2 and 3 by Hg2+, Mn2+, Co2+, Cd2+ and Cs+ respectively. The denat uration constants calculated for the luciferase-metal ion complexes vary be tween 0.152 and 0.570 and follow the order of Hg2+>Cs+>Cd2+>Co2+>Mn2+. Stea dy state fluorescence data reveal that the metal ions quench the fluorescen ce of enzyme by complexation with the side chain residues of the excited st ate tyrosine. Profound change in the UV CD spectrum of luciferin and lucife rase in presence of metal ions was attributed to the conformational change in the substrate and enzyme. Thus the inhibition of luciferase activity in the firefly bioluminescence by metal ions is attributed to the quaternary c omplex formed between metal ion-luciferin-luciferase and ATP near or around the active site of the enzyme.