Application of 2D fluorescence correlation method to investigate the dilution-induced heterogeneous distribution of the bound FMN in azoreductase

AzoR is a homodimeric, flavin mononucleotide (FMN)-containing, NADH-dependent azoreductase from Escherichia coli. In this paper, we investigated the effect of the concentration of both AzoR and R59G on the spectral behavior of the bound FMN using two-dimensional fluorescence correlation spectra. Two cross peaks (530, 490) and (580, 530) were observed from the dilution-induced 2D asynchronous correlation map of wt AzoR, while only one cross peak appeared at (600, 530) for R59G mutant. This result indicated that the mutation at site 59 influenced the formation of dilution-induced intermediates. The specific activity of both AzoR and R59G mutant was unaffected by dilution when the enzyme concentration is below 1 μmol/L, which suggested that no significant dissociation of FMN occurred at low concentrations. Additionally, in order to explore the origin of these intermediates, we carried out a 2D correlation analysis using excitation wavelength-dependent fluorescence emission spectroscopy. The results showed that there coexisted two types of FMN that emitted fluorescence at 530 nm and 500 nm, respectively. Taken together, these results suggested that the 2D method is a very powerful method to identify the heterogeneous distribution of the bound FMN in solution.

Graphical abstractDilution-induced two-dimensional synchronic and asynchronic fluorescence emission spectral changes of wt AzoR (A and D), R59G mutant (B and E) and free FMN (C and F) in 20 mmol/L Tris–HCl (pH 7.4) at room temperature upon excitation at 450 nm.Figure optionsDownload full-size imageDownload as PowerPoint slide