Firefly luciferase catalyzes the highly efficient emission of yellow-green light from substrate luciferin by a series of reactions that require MgATP and molecular oxygen. We prepared 2-(4-benzoylphenyl)thiazole-4-carboxylic acid (BPTC), a novel benzophenone-based substrate analog, intending to use it in photoaffinity labeling studies to probe the luciferase active site. Instead, we found that while BPTC was a potent photoinactivating reagent for firefly luciferase, it was not a photoaffinity labeling agent. Using proteolysis, reverse phase high-performance liquid chromatography, tandem high performance liquid chromatography-electrospray ionization mass spectrometry, and Edman sequencing, we identified a single luciferase peptide, 244HHGF247, the degradation of which was directly correlated to luciferase photoinactivation. Results of enzyme kinetics and related studies were consistent with this peptide being at or near the luciferin binding site. Further, peptide model studies and additional investigations on the nature of the photoinactivation process strongly suggested that BPTC catalyzed the formation of singlet oxygen at the active site of the enzyme. We describe here an uncommon example of active site-directed photooxidation of an enzyme by singlet oxygen.Bioluminescence is a captivating process in which living organisms convert chemical energy into light. Beetle bioluminescence, including perhaps 2000 species of fireflies (1, 2), has provided a fertile area of basic and applied research mainly focused on the North American firefly Photinus pyralis. As Equations 1 and 2 indicate, the key firefly luciferase enzyme functions as a monooxygenase, although it does so without the apparent involvement of a metal or cofactor. By some means, amino acid residues are recruited to promote the addition of molecular oxygen to luciferin which is then transformed to an electronically excited state oxyluciferin molecule and CO 2 , each containing one oxygen atom from molecular oxygen. The light emission process is extremely efficient; nearly a single photon is emitted per reacted luciferin molecule (3).The luciferase-catalyzed reactions provide the basis for a wide variety of biochemical assays, many with important clinical applications (4, 5). Moreover, the luciferase gene (6) is widely used as a reporter in studies of gene expression and regulation (7). Additionally, the cloning and sequencing of P. pyralis luciferase and homologues from several beetles (8) have revealed that these enzymes are closely related to a large family of nonbioluminescent proteins (9, 10) that catalyze reactions of ATP with carboxylate substrates to form acyl adenylates.The recent solving of the crystal structure of luciferase without bound substrates or ligands revealed an apparently unique molecular architecture consisting of two distinct domains (11); however, a detailed description of the luciferase active site remains elusive. Early chemical modification studies have failed to document the involvement of any specific amino acid residues in s...