Isomers in the excited state of electron-transferring flavoprotein from Megasphaera elsdenii: Spectral resolution from the time-resolved fluorescence spectra

Electron-transferring flavoprotein (Holo-ETF) from Megasphaera elsdenii contains two FAD’s, one of which easily dissociates to form Iso-ETF (contains one FAD). Time-resolved fluorescence of FAD in Iso-ETF, and Holo-ETF were measured at 5 °C and 25 °C. Wavelength-dependent fluorescence decays of the both ETF at 5 °C and 25 °C were analyzed to resolve them into two independent spectra. It was found that Iso-ETF displayed two spectra with lifetime of 0.605 ns (emission peak, 508 nm) and with lifetime of 1.70 ns (emission peak, 540 nm) at 5 °C, and with lifetime of 0.693 ns (emission peak, 508 nm) and with lifetime of 2.75 ns (emission peak, 540 nm) at 25 °C. Holo-ETF displayed two spectra with lifetime of 0.739 ns (emission peak, 508 nm) and with lifetime of 2.06 ns (emission peak, 545 nm) at 5 °C, and with lifetime of 0.711 ns (emission peak, 527 nm) and with lifetime of 3.08 ns (emission peak, 540 nm) at 25 °C. Thus fluorescence lifetimes of every spectrum increased upon elevating temperature. Emission peaks Iso-ETF did not change much upon elevating temperature. Activation enthalpy changes, activation entropy changes and activation Gibbs energy changes of quenching rates all displayed negative. Two emission species in the both ETF may be hydrogen-bonding isomers, because isoalloxazine ring of FAD contains four hydrogen acceptors and one donor.