title: Decomposition of the fluorescence spectra of two FAD molecules in electron-transferring flavoprotein from Megasphaera elsdenii.
abstract: 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.
authors.affiliation: Department of Molecular Physiology, Faculty of Life Sciences, Kumamoto University, 1-1-1 Honjo, Chuo-ku, Kumamoto, Kumamoto 860-8556, Japan. satok@kumamoto-u.ac.jp
authors.forename: Kyosuke
authors.initials: K
authors.lastname: Sato
authors.forename: Yasuzo
authors.initials: Y
authors.lastname: Nishina
authors.forename: Kiyoshi
authors.initials: K
authors.lastname: Shiga
mesh_terms: D000249:Adenosine Monophosphate / Q000737:chemistry; D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry*; D044927:Electron-Transferring Flavoproteins / Q000737:chemistry*; D005182:Flavin-Adenine Dinucleotide / Q000737:chemistry*; D005415:Flavins / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D045851:Megasphaera / Q000737:chemistry*; D011487:Protein Conformation; D013045:Species Specificity; D013050:Spectrometry, Fluorescence; D013552:Swine
chemical_list: D001426:Bacterial Proteins; D044927:Electron-Transferring Flavoproteins; D005415:Flavins; D005182:Flavin-Adenine Dinucleotide; D000249:Adenosine Monophosphate
keywords: Megasphaera elsdenii; electron-transferring flavoprotein; excitation-emission matrix of fluorescence; singular value decomposition; unusual absorption spectrum