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

作　　者：Juan Feng Ting Han Mi-Qing Zhang Yu Zhou Qing-Qin Wu 

机构地区：[1]School of Life Science and Technology,University of Electronic Science and Technology of China

出　　处：《Chinese Chemical Letters》2015年第2期210-214,共5页中国化学快报（英文版）

基　　金：fund by sub-project of National Science and Technology Major Project on Water Pollution Prevention and Control(No.2012ZX07203-003-Z04);supported by the Fundamental Research Funds for the Central Universities(No. ZYGX2012J112)

摘　　要：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 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.

关 键 词：Azoreductase FMN 2D fluorescence correlation spectra Heterogeneous distribution 

分 类 号：O629.8[理学—有机化学] O657.3[理学—化学]