Mapping the structural dynamics of red- and blue-emitting beetle luciferases revealed by HDX-MS

Beetle luciferases catalyze bioluminescent reactions using identical substrates-luciferin, ATP, and oxygen-yet emit colors ranging from green to red. As the luciferase fold is highly conserved, the structural basis of this spectral diversity remains elusive. Hydrogen/deuterium exchange mass spectrometry (HDX-MS) was used to systematically probe the conformational dynamics of green-emitting <i>Amydetes vivianii</i> luciferase (GB_Av) and red-emitting <i>Phrixothrix hirtus</i> luciferase (RE_Ph), along with their color-shifting mutants, R337L and L334R, respectively. HDX-MS profiling in the apo and ligand-bound states mapped dynamic changes associated with substrate binding and color modulation. RE_Ph and the red-shifted R337L GB_Av mutant exhibited globally higher dynamics, particularly in N-terminal domain peptides surrounding the active site, than their blue-green-emitting counterparts. Substrate binding reduced deuterium uptake in all enzymes; however, red-emitting forms retained greater flexibility. These findings establish a direct link between structural dynamics and bioluminescence color tuning in beetle luciferases, providing mechanistic insight into spectral control.