Abstracts

Bioluminescence is the emission of light from a biological process, and it can be found in various organisms, including bacteria, fireflies, and beetles. Luciferase is an enzyme that catalyzes a two-stage chemical reaction to oxidize luciferin in the presence of Mg²⁺ and ATP to produce oxyluciferin and release energy in the form of visible light. Previously, we determined the crystal structures of two beetle luciferases with red and blue-shifted green light emission^[1]. The blue-shifted green-emitting luciferase from the firefly Amydetes vivianii (GB_Av) and the naturally red-emitting luciferase from the glow-worm Phrixothrix hirtus (RE_Ph) that was found as tetramers or octamers in solution as well as in the crystal lattice^[1]. To understand the protein dynamics during the emission of light, multiple mutations were introduced in the dimer and tetramer interfaces. The loop^346‒361 present at the bottom of the active site was found to be important in the blue- and red-emitting luciferases, where the green emission of GB_Av was shifted from 539 nm to 580 nm, and the red emission of RE_Ph was shifted from 623 nm to 603 nm. To better understand the conformational dynamic associated with the different color emission for the wild-type WT and mutant enzymes of GB_Av and RE_Ph, hydrogen-deuterium exchange with mass spectrometry (HDX-MS) measurements were used to study the protein dynamics at different conformational states induced by the binding of the product, oxyluciferin. High dynamics and deuterium exchange rates have been associated with the apo-state compared to the oxyluciferin bound-state. In addition, a comparative analysis is conducted on the WT enzyme and the mutants that red-shifted the green emission of GB_Av or blue-shifted the red emission of RE_Ph. The HDX-MS data reveal high dynamics on the loop^346‒361 for the GB_Av, while less dynamic variation was observed in the RE_Ph. Also, the rate of deuterium labeling increased for the GB_Av mutants with red-shifted emission compared to the WT RE_Ph. In contrast, the WT GB_Av was more stable at half of the rate of dynamics compared to the WT RE_Ph.