Electrostatic interactions between firefly oxyluciferin and the surrounding proteins were analyzed, and the amino acids important for controlling emission energy were identified. We propose Arg223Ala, Glu344Ala, and Asp422Ala mutations in firefly oxyluciferase of Photinus pyralis, which artificially change the luminescence color by tuning the electrostatic effect from the luciferase proteins. In the theoretical mutation simulation, the emission energy of the triple mutant was estimated to be 2.05 eV ( 602 nm, reddish-orange), which is 0.18 eV lower than that of the wild type ( 2.23 eV, 557 nm, yellow-green). For calculating the emission energies, we used the symmetry-adapted cluster-configuration interaction (SAC-CI) method. (C) 2008 Elsevier B. V. All rights reserved.