Microelectrospray ionization analysis of noncovalent interactions within the electron transferring flavoprotein.
Cofactor associations within the electron transferring flavoprotein (ETF) were studied in real time using microelectrospray ionization-mass spectrometry (muESI-MS). Initial analysis of porcine (pETF) and human ETF (hETF) revealed only the holoprotein. When muESI-MS source energies were increased, both pETF and hETF readily lost AMP. Analysis of hETF and pETF in methanol revealed intact alpha- and beta-subunits, and beta-subunit with AMP. The pETF also contained beta-subunit with FAD and beta-subunit with both cofactors. In contrast to crystal structure predictions, AMP dissociates more readily than FAD, and the pETF beta-subunit has an intimate association with FAD. This work demonstrates the complementarity of muESI-MS with NMR X-ray and optical spectroscopy in the analysis of noncovalent complexes.
Hoard
H M
HM
Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, Minnesota, 55905
Benson
L M
LM
Vockley
J
J
Naylor
S
S
D000818:Animals; D044927:Electron-Transferring Flavoproteins; D005420:Flavoproteins / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D008099:Liver / Q000737:chemistry; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D013552:Swine
D044927:Electron-Transferring Flavoproteins; D005420:Flavoproteins