Decomposition of the fluorescence spectra of two FAD molecules in electron-transferring flavoprotein from Megasphaera elsdenii

• Published in: Journal of biochemistry (Tokyo) 2013-07, Vol.154 (1), p.61-66
• Main Authors: Sato, Kyosuke, Nishina, Yasuzo, Shiga, Kiyoshi
• Format: Article
• Language: English
• Subjects: Adenosine Monophosphate - chemistry; Animals; Bacterial Proteins - chemistry; Electron-Transferring Flavoproteins - chemistry; Flavin-Adenine Dinucleotide - chemistry; Flavins - chemistry; Hydrogen-Ion Concentration; Megasphaera - chemistry; Protein Conformation; Species Specificity; Spectrometry, Fluorescence; Swine
• ISSN: 0021-924X; 1756-2651
• DOI: 10.1093/jb/mvt027

Electron-transferring flavoprotein (ETF) from Megasphaera elsdenii contains two FAD molecules, FAD-1 and FAD-2. FAD-2 shows an unusual absorption spectrum with a 400-nm peak. In contrast, ETFs from other sources such as pig contain one FAD and one AMP with the FAD showing a typical flavin absorption spectrum with 380- and 440-nm peaks. It is presumed that FAD-2 is the counterpart of the FAD in other ETFs. In this study, the FAD-1 and FAD-2 fluorescence spectra were determined by titration of FAD-1-bound ETF with FAD using excitation-emission matrix (EEM) fluorescence spectroscopy. The EEM data were globally analysed, and the FAD fluorescence spectra were calculated from the principal components using their respective absorption spectra. The FAD-2 fluorescence spectrum was different from that of pig ETF, which is more intense and blue-shifted. AMP-free pig ETF in acidic solution, which has a comparable absorption spectrum to FAD-2, also had a similar fluorescence spectrum. This result suggests that FAD-2 in M. elsdenii ETF and the FAD in acidic AMP-free pig ETF share a common microenvironment. A review of published ETF fluorescence spectra led to the speculation that the majority of ETF molecules in solution are in the conformation depicted by the crystal structure.