Bioluminescence Color Determinants of Phrixothrix Railroad‐worm Luciferases: Chimeric Luciferases, Site‐directed Mutagenesis of Arg 215 and Guanidine effect ¶

Chimeric proteins were produced using the green light–emitting luciferase of Phrixothrix vivianii (PxGr: λ max = 548 nm) and the red light–emitting luciferase of Phrixothrix hirtus (PxRe: λ max = 623 nm). Constructs containing residues 1–344 of the red light–emitting luciferase with residues 345–545 of the green light emitting one emitted red light (PxReGr; λ max = 613 nm), while the reverse emitted green light (PxGrRe; λ max = 552 nm). From these results we conclude that the region 1–344 determines the color of bioluminescence (BL) in railroad‐worm luciferases, and that residues above 344 are not involved. The substitution R215S in the green light–emitting luciferase (PxGr) resulted in a ∼40 nm redshift on the BL spectrum (λ max = 585 nm) and an associated decrease of activity, whereas the same mutation in PxRe luciferase had little effect. Guanidine was shown to cause blueshifts in the BL spectra and stimulate the activity of the red‐emitting luciferases (from λ max = 623 to λ max = 600 nm) and in PxGr R215S (from λ max = 585 to λ max = 560 nm) mutant luciferase, but not in the green‐emitting luciferases, suggesting that guanidine can simulate positively charged residues involved in BL color determination.