Identifications of the true carbon-13 nuclear magnetic resonance spectrum of the stable intermediate II in bacterial luciferase

Intermediate II in bacterial luciferase, formed in a reaction of luciferase with FMNH super(-) and O sub(2) has been reinvestigated by super(13)C NMR spectroscopy with super(13)C-enriched FMN derivatives. It is shown that the previously published spectrum of the intermediate by Ghisla et al. (Ghisla, S., Hastings, J.W., Favaudon, V., & Lhoste, J.-M. (1978) Proc. Natl. Acad. Sci., USA 75, 5860-5863) does not represent the intermediate but is due to a contamination contained in commercially available ( super(2)H sub(6))ethylene glycol. Relaxation studies show that the resonance line due to the byproduct of ( super(2)H sub(6))ethylene glycol is easier to observe at low temperature than at room temperature, explaining fully the spectral properties of the published artifact. The true C(4a) resonance is found at 82.5 ppm, and this is interpreted as due to considerable sp super(2) hybridization at this position, indicating an almost planar configuration by comparison with a model compound.