Among beetle luciferases, the pH-sensitive firefly luciferases have been studied extensively. Much less is known about pH-insensitive luciferases, which include click beetle and railroad worm luciferases. Previously, we found that the residues R215 and T226 (N230) are important for green light emission. Here we show that the conserved residue A243 in pH-insensitive luciferases and the corresponding G247 in pH-sensitive luciferases affect the emission spectrum and influence pH-sensitivity. In contrast to railroad worm green light-emitting (PxvGR) and firefly luciferases, the substitution of R215 in Pyrearinus termitilluminans click beetle luciferase (Pte) had no effect on the spectrum, showing that R215 is not essential for green light emission in all beetle luciferases. A homology-based model of Pte luciferase shows that R215 and T226 are close enough to interact. To investigate if there was an interaction between these conserved residues, double mutants were constructed. The double substitution R215S/T226N in Pte luciferase abolished the activity. In PxvGR luciferase the same double mutant resulted in a redshift (lambda(max) = 595 nm), whose magnitude was lower than the value expected for an additive effect. These results suggest that the effects of R215S and T226N are partially interdependent. The double substitution T226N/A243G had an additive redshift effect on the spectrum of PxvGR luciferase, whereas it had a smaller effect on the spectrum of Pte luciferase. Altogether, these results suggest that the above substitutions have different effects on the active site of click beetle and railroad worm luciferases.