Chemical Synthesis of a Copepod-Derived Artificial Luciferase and Characterization of Site-specific Oxidation During Bioluminescence

Luciferases are enzymes that catalyze bioluminescent reactions: they are used widely for bioimaging and in reporter systems. Among them, copepod luciferases have distinct structures and bioluminescence profiles, but their structure(s) and mechanism(s) remain poorly understood. Chemical protein synthesis enables precise structural and functional analyses using homogeneous samples with site-specific modifications. In this study, we first investigated the chemical synthesis of picALuc, a minimal artificial luciferase derived from copepod luciferases. picALuc was successfully synthesized through sequential native chemical ligations and subsequent oxidative refolding. During analysis of the bioluminescence reaction of synthetic picALuc, we unexpectedly observed the generation of a mono-oxygenated form of the protein. To characterize the oxidation site and its impact on the luminescence properties, we designed and synthesized several picALuc variants in which the methionine residues of the protein (Met41 and Met82) were replaced with either norleucine or methionine sulfoxide. Experiments revealed that oxidation of picALuc occurred specifically at Met82. The oxygenation did not affect the rapid luminescence-decay (i.e., the “flash-type”) kinetics of picALuc. Comparative analysis of methionine sulfoxide-containing variants revealed that the position of methionine sulfoxide differentially affects the protein folding efficiency and overall hydrophilicity. These results provide valuable insights for the development of miniature luciferases with prolonged bioluminescence suitable for bioimaging applications.