Formation of Coelenteramine from 2-Peroxycoelenterazine in the Ca2+-Binding Photoprotein Aequorin

Aequorin consists of apoprotein (apoAequorin) and (S)-2-peroxycoelenterazine (CTZ-OOH) and is considered to be a transient-state complex of an enzyme (apoAequorin) and a substrate (coelenterazine and molecular oxygen) in the enzymatic reaction. The degradation process of CTZ-OOH in aequorin was characterized under various conditions of protein denaturation. By acid treatment, the major product from CTZ-OOH was coelenteramine (CTM), but not coelenteramide (CTMD), and no significant luminescence was observed. The counterparts of CTM from CTZ-OOH were identified as 4-hydroxyphenylpyruvic acid (4HPPA) and 4-hydroxyphenylacetic acid (4HPAA) by liquid chromatography/electrospray ionization-time-of-flight mass spectrometry (LC/ESI-TOF-MS). In the luminescence reaction of aequorin with Ca2+, similar amounts of 4HPPA and 4HPAA were detected, indicating that CTM is formed by two pathways from CTZ-OOH through dioxetanone anion and not by hydrolysis from CTMD.

Automated summary

This study investigated the degradation process of CTZ-OOH in aequorin and found that CTM was the major product after acid treatment, with no significant luminescence. 4HPPA and 4HPAA were identified as the counterparts of CTM in CTZ-OOH using mass spectrometry. The luminescence reaction of aequorin with Ca2+ showed similar amounts of 4HPPA and 4HPAA, suggesting that CTM is formed through dioxetanone anion rather than hydrolysis from CTMD.