Abstract
Bacterial luciferase catalyzes a bioluminescent oxidation of reduced flavin mononucleotide; the products include a photon and oxidized FMN. The experiments reported here show that luciferase binds oxidized flavin mononucleotide in a 1:1 molar ratio with an apparent dissociation constant of 1.2 times 10-4 M at 3 degrees in 0.05 M 2,2-bis(hydroxymethyl)-2,2'2"-nitriloethanol (bis-tris), pH 7.0. Analysis of the binding at temperatures between 3 and 30 degrees indicates an enthalpy of binding (delta H a) of minus 10.0 kcal per mol. The absorption spectrum of luciferase-bound FMN shows considerable alteration relative to that of free flavin. There is one major peak at 366 nm, and the 445-nm band is resolved into two distinct peaks at 434 and 458 nm; this spectrum is indicative of binding in a nonpolar environment. The circular dichroism spectrum of FMN bound to luciferase has structure which correlates well with the optical absorption spectrum of the bound flavin. The detail in the spectra of the bound FMN probably reflects the resolution of vibrational structure which is blurred in polar environments. The optical activity shown by the CD spectrum presumably results from binding in an electronically asymmetric fashion. Although FMN free in solution is highly fluorescent, FMN bound to luciferase is nonfluorescent, thus indicating that the emitting species is not an excited state of product FMN located in the same site in which luciferase binds oxidized FMN.
Highlights
Since the bioluminescent quantum yield with respect to flavin is about 0.05 to 0.15 [12, 19], the results indicate that the emission in the bioluminescent reaction does not occur from the excited state of product FMN located in the same site in which luciferase binds oxidized FMN
FMN-The most striking feature of the optical absorption spectrum of FMN bound to bacterial luciferase is its highly resolved structure (Fig. la)
In a previous report a lower limit for the dissociation constant of FMN and luciferase was estimated to be 1 x 1w4 M, at least 3 orders of magnitude greater than that estimated for FMNHr, the substrate [29]
Summary
Materials-FMN and bis-tris were products of Sigma. The FMN, stored desiccated at -2O”, was estimated from thin layer chromatography in a variety of solvent systems to be more than 90%pure. Materials-FMN and bis-tris were products of Sigma. The FMN, stored desiccated at -2O”, was estimated from thin layer chromatography in a variety of solvent systems to be more than 90%. This level of purity is confirmed by the homogeneity of binding to luciferase observed in these experiments. FMN concentrations were determined spectrophotometrically on the basis of a molar absorption coefficient of 12,200 M-l cm-l at 450 nm [20]. All binding studies were done in 0.05 M bis-tris-HCl, pH 7.0. Buffers for use in equilibrium dialysis contained, in addition, 0.2 M NaCl
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