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

• Published in: Chinese chemical letters Vol. 26; no. 2; pp. 210 - 214
• Main Authors: Feng, Juan, Han, Ting, Zhang, Mi-Qing, Zhou, Yu, Wu, Qing-Qin
• Format: Journal Article
• Language: English
• Published: Elsevier B.V 01.02.2015
• Subjects: 2D fluorescence correlation spectra; Azoreductase; FMN; Heterogeneous distribution; 不均匀分布; 二维; 偶氮还原酶; 应用; 相关方; 稀释; 荧光发射光谱; 黄素单核苷酸; Heterogeneous distribution; FMN; Azoreductase; 2D fluorescence correlation spectra
• ISSN: 1001-8417; 1878-5964
• DOI: 10.1016/j.cclet.2014.11.019

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.