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

Juan Feng ,  Ting Han ,  Mi-Qing Zhang ,  Yu Zhou ,  Qing-Qin Wu

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Citation:  Juan Feng, Ting Han, Mi-Qing Zhang, Yu Zhou, Qing-Qin Wu. Application of 2D fluorescence correlation method to investigate the dilution-induced heterogeneous distribution of the bound FMN in azoreductase[J]. Chinese Chemical Letters, 2015, 26(2): 210-214. doi: 10.1016/j.cclet.2014.11.019 shu

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

    • 通讯作者: Juan Feng,
  • 基金项目:

    Control (No. 2012ZX07203-003-Z04)  (No. 2012ZX07203-003-Z04)

    supported by the Fundamental Research Funds for the Central Universities (No. ZYGX2012J112). (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 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 mmol/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.

English

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  • 发布日期:  2014-11-20
  • 收稿日期:  2014-09-01
  • 网络出版日期:  2014-10-22
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