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 R59 G 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 R59 C mutant.This result indicated that the mutation at site 59 influenced the formation of dilution-induced intermediates.The specific activity of both AzoR and R59 G 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 a2 D 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. 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 correlation map of wt AzoR, while only one cross peak was at (600,530) for R59 C mutant. at site 59 influenced the formation of dilution-induced intermediates. The specific activity of both AzoR and R59 G 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 a2 D correlation analysis using excitation wavelength-dependent fluorescence emission spectroscopy. Results showe d 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.