AbstractCa2+‐regulated photoproteins use a noncovalently bound 2‐hydroperoxycoelenterazine ligand to emit light in response to Ca2+ binding. To better understand the mechanism of formation of active photoprotein from apoprotein, coelenterazine and molecular oxygen, we investigated the spectral properties of the anaerobic apo‐obelin–coelenterazine complex and the kinetics of its conversion into active photoprotein after exposure to air. Our studies suggest that coelenterazine bound within the anaerobic complex might be a mixture of N7‐protonated and C2(−) anionic forms, and that oxygen shifts the equilibrium in favor of the C2(−) anion as a result of peroxy anion formation. Proton removal from N7 and further protonation of peroxy anion and the resulting formation of 2‐hydroperoxycoelenterazine in obelin might occur with the assistance of His175. It is proposed that this conserved His residue might play a key role both in formation of active photoprotein and in Ca2+‐triggering of the bioluminescence reaction.