Fig. 5: Catalytic mechanism for NanoLuc-type luciferase reaction.

a 2Fo-Fc electron density map (contour level 3.0 σ) of azaCTZ bound in the intra-barrel catalytic site of NanoLuc^CTZ. b Cartoon representation of the overall structure of NanoLuc^CTZ (cyan) with bound azaCTZ (green). c Close-up view of azaCTZ (green) bound to NanoLuc^CTZ, with important residues creating the active site in cyan stick representation. Red sphere; a water molecule bound in the catalytic site. Key molecular contacts are shown as dashed yellow lines. d Mutagenesis of NanoLuc active site residues. Data indicate the average relative luciferase activities of each mutant. Assays were done in triplicate; error bars represent standard deviations. e Visualizations of the NanoLuc^CTZ catalytic site with modeled CTZ (i), 2-peroxy-CTZ (ii), CTZ dioxetanone (iii), and CEI (iv). Key protein residues are shown as sticks and lines, molecular oxygen (OXY) is shown in red sphere, and hydrogen bonds are shown as dashed lines. f A blueprint for NanoLuc-type reaction mechanism. The cycle starts with the binding of CTZ into the catalytic site localized inside the β-barrel structure, and it enters with a deprotonated imidazopyrazinone core, as demonstrated experimentally in previous work²¹. I. Upon the entry, the -OH group of the C6-(p-hydroxyphenyl) moiety is hydrogen-bonded with two tyrosines (Y94 and Y114), as well as deprotonated by D139, yielding the activated dianion O10-CTZ. Then, the side chain of R162, and perhaps helped by the side chain of Q42, position a co-substrate molecule (dioxygen) such that it can be attacked by the C2 carbon of O10-CTZ. II. The initial interaction proceeds via a charge-transfer radical mechanism. III. The next steps encompasses radical pairing and termination, resulting in the 2-peroxy-CTZ anion, which then undergoes intramolecular cyclization via a nucleophilic addition-elimination mechanism. IV. This cyclization generates a dioxetanone intermediate with a deprotonated amide group. V. The side chain of R162 protonates the amide group of oxyluciferin in order to avoid attenuation of the luminescence. VI. The energy-rich dioxetanone intermediate is unstable and decomposes by decarboxylation into an excited CEI product. VII. When returning to the ground state, the excited CEI releases a blue photon. Finally, the protonation status of R162 is restored by the proton transfer from a water molecule.