Chemical Mechanism of Bioluminescence in <i>Diplocardia longa</i> : A Combined Theoretical and Experimental Investigation

<i>Diplocardia longa</i> (<i>D. longa</i>), a bioluminescent earthworm native to North America, emits blue-green light. Experimental studies conducted in the 1970s-1980s established that its bioluminescence (BL) involves reactions of H₂O₂, luciferin (<b>L</b>) and Cu⁺-dependent luciferase. However, research stalled after 1981, leaving some fundamental gaps: the chemical mechanism, key intermediates, product, light emitter, and role of Cu⁺ remain undefined. To address these uncertainties, this combined theoretical and experimental study systematically investigated the corresponding chemiluminescence (CL) of <i>D. longa</i> BL, since the crystal structure of luciferase has not yet been determined. Using density functional theory (DFT) and mixed reference spin flip (MRSF) DFT methods, we calculated the complete reaction pathway from reactants to light emission. Integrated with experimental evidence, we confirm the catalytic function of Cu⁺, identify the molecular structure of the peroxide intermediate and product, and assign the light emitter. This study provides critical insights into natural BL systems, particularly as <i>D. longa</i> luciferin is a shared substrate across 13 bioluminescent earthworm species spanning six genera.