Crystal Structure of Obelin after Ca²⁺-Triggered Bioluminescence Suggests Neutral Coelenteramide as the Primary Excited State

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 103; no. 8; pp. 2570 - 2575
• Main Authors: Liu, Zhi-Jie, Stepanyuk, Galina A., Vysotski, Eugene S., Lee, John, Markova, Svetlana V., Malikova, Natalia P., Wang, Bi-Cheng
• Format: Journal Article
• Language: English
• Published: United States National Academy of Sciences 21.02.2006; National Acad Sciences
• Subjects: Amides - chemistry; Animals; Anions; Atoms; Binding Sites; Biochemistry; Biological Sciences; Bioluminescence; Calcium; Calcium - chemistry; Crystal structure; Crystallography; Hydrogen; Hydrogen bonds; Ions; Luminescence; Luminescent proteins; Luminescent Proteins - chemistry; Luminescent Proteins - genetics; Molecules; Mutation; Neutrality; Oxygen; Protein Conformation; Proteins
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.0511142103

The crystal structure at 1.93-Å resolution is determined for the $Ca^{2+}$-discharged obelin containing three bound calcium ions as well as the product of the bioluminescence reaction, coelenteramide. This finding extends the series of available spatial structures of the ligand-dependent conformations of the protein to four, the obelin itself, and those after the bioluminescence reaction with or without bound $Ca^{2+}$ and/or coelenteramide. Among these structures, global conformational changes are small, typical of the class of "calcium signal modulators" within the EF-hand protein superfamily. Nevertheless, in the active site there are significant repositions of two residues. The His-175 imidazole ring flips becoming almost perpendicular to the original orientation corroborating the crucial importance of this residue for triggering bioluminescence. Tyr-138 hydrogen bonded to the coelenterazine Nl-atom in unreacted obelin is moved away from the binding cavity after reaction. However, this Tyr is displaced by a water molecule from within the cavity, which now forms a hydrogen bond to the same atom, the amide N of coelenteramide. From this observation, a reaction scheme is proposed that would result in the neutral coelenteramide as the primary excited state product in photoprotein bioluminescence. From such a higher energy state it is now energetically feasible to account for the shorter wavelength bioluminescence spectra obtained from some photoprotein mutants or to populate the lower energy state of the phenolate anion to yield the blue bioluminescence ordinarily observed from native photoproteins.