Abstract
Firefly luciferases produce yellow-green light under physiological and alkaline conditions, however at acidic pH, higher temperatures or in the presence of heavy metals the color changes to red, a property called pH-sensitivity. Despite many decades of studies, the proton and metal binding sites responsible for pH-sensitivity remain enigmatic. Previously we suggested that the salt bridge E311/R337 keeps a closed conformation of the luciferin phenolate binding site. Here we further investigated the effect of this salt bridge and mutations of the neighbor residues H310 and E/N354, on metal and pH-sensitivity of firefly luciferases emitting distinct bioluminescence colors (Cratomorphus distinctus: 548 nm; Macrolampis sp2: 569 nm). The substitutions of H310 and E/N354 modulate metal sensitivity, whereas the carboxylate of E311 may work as the catalytic base essential for green bioluminescence and pH-sensitivity. Modeling studies showed that H310, E311 and E354 side-chains coordinate Zinc, constituting the metal binding site and the pH-sensor. Electrostatic potential and pKa calculations suggest that the external couple H310/E354 is affected by pH, whereas E311/R337 make a stabilized internal pair which retains excited oxyluciferin ejected proton near its phenolate group into a high energy state, promoting yellow-green bioluminescence. Protonation or metal binding weaken these electrostatic gates and their ability to retain the excited oxyluciferin released proton near its phenolate, promoting red light emission.
Highlights
Luciferases are the enzymes which elicit the beautiful yellow-green flashes of fireflies during summer nights around the word
The three-dimensional structures have been solved for the North-American firefly luciferase Photinus pyralis (Ppy) in the absence of substrates[36], in the presence of bromophorm[37] and with the C-terminal trapped in a closed conformation[38], for the Japanese L. cruciata firefly luciferase complexed with the luciferyl-adenylate analogue DLSA in a closed conformation and with oxyluciferin and AMP in an open conformation[39], and for Lampyris turkanensis luciferase[40]
H245 is invariant and H244 is conserved in beetle luciferases displaying distinct bioluminescence colors, none of them could play the role of basic residues assisting tautomerization of excited oxyluciferin[13]
Summary
Luciferases are the enzymes which elicit the beautiful yellow-green flashes of fireflies during summer nights around the word They catalyze an ATP-dependent oxidation of a benzothiazolic luciferin[1,2]. The presence of bases near the thiazinic side of the luciferin binding site assisting the tautomerization between a keto and enol forms has been originally claimed to explain green to red bioluminescence color change in firefly luciferases[9,13]. Mutation of the luciferin binding site residues, including histidines H244 and H245, which supposedly could assist oxyluciferin tautomerization in the thiazolyl part of the active site, resulted in red mutants in firefly luciferases[41,42,43]. Interactions of main-chain amide bonds with oxyluciferin phenolate[44] and the active site conformation, were proposed to be important for bioluminescence colors
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