Abstracts

Beetle luciferases are responsible for catalyzing the oxidation of luciferin producing bioluminescence colors ranging from green to red, with unique properties. Fireflies emit yellow-green light, click beetles green to orange light depending on the lanterns, and railroad worms from green to red depending on the lantern. Furthermore, firefly luciferases can modulate bioluminescence colors from green to red depending on the pH, presence of heavy metals and temperature. Despite decades of structural/functional studies, mainly with firefly luciferases, how luciferases determine and modulate BL colors, remain an elusive issue that physicists, chemists, biochemists and molecular biologists have tried to solve. Our group has cloned the largest variety of beetle luciferases, including pH-sensitive and pH-insensitive which naturally producing green-blue, green, yellow-green, yellow, orange and red from distinct families. Through comparative studies, site-directed mutagenesis, modelling and biophysical methods like circular dichroism, our results indicate that green-yellow light emission could be obtained through: (1) a single rigid and hydrophobic active site conformation or (2) by pH-modulated rigidification in firefly luciferases during the emitting step. On the other hand, red light emission is obtained through a lousier active site conformation, especially a larger oxyluciferin binding site pocket, during the emissive step. Altogether the results indicate that oxyluciferin phenolate binding pocket rigidity modulate excited state proton transfer and electrostatic interactions, decreasing or increasing energy levels of excited and ground states, thereof modulating bioluminescence colors. The results also opened the possibility to develop Far Red emitting combinations by combining luciferase engineering and luciferin analogs, and color tuning luciferases por pH and toxic metal biosensing (Financial support: FAPESP 2022/04800-0; CNPq 401.050/2021-1)