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

Abstract

256-267D(-) Luciferin, interacts with different metal ions to produce colourless soluble salts with absorption spectra broader, intense and red shifted as compared to those of the parent compound. The equilibrium constants for the luciferin-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 lucifcrin and luciferase are larger than that of binary complexes but vary in the same order. Steady state fluorometric titration's of luciferin further confirmed its complexation with metal ions. The single absorption maximum of firefly luciferase at 278 nm originating from tyrosine was split into a doublet in presence of transition metal ions. The absorption maximum at lower wavelength is attributed to the H-bond raptured free tyrosine denatured conformation of the luciferase while the longer wavelength band to tyrosine- transition metal ion complex. Difference spectra of luciferase metal ion complex yielded change in the molar extinction coefficients from which the number of tyrosine molecules exposed to aqueous solution by the perturbant metal ions are evaluated following the Donovan model. The number of tyrosine molecules exposed to 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 denaturation constants calculated for the luciferase-metal ion complexes vary between 0.152 and 0.570 and follow the order of Hg2+> Cs+> Cd2+> Co2+> Mn2+. Steady state fluorescence data reveal that the metal ions quench the fluorescence of enzyme by complexation with the side chain residues of the excited state tyro sine. Profound change in the UV CD spectrum of luciferin and luciferase 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 complex formed between metal ion-luciferin-luciferase and ATP near or around the active site of the enzyme