From the outset, canonical electron transferring flavoproteins (ETFs) earned a reputation for containing modified flavin. We now show that modification occurs in the recently recognized bifurcating (Bf) ETFs as well. In Bf ETFs, the 'electron transfer' (ET) flavin mediates single electron transfer via a stable anionic semiquinone state, akin to the FAD of canonical ETFs, whereas a second flavin mediates bifurcation (the Bf FAD). We demonstrate that the ET FAD undergoes transformation to two different modified flavins by a sequence of protein-catalyzed reactions that occurs specifically in the ET site, when the enzyme is maintained at pH 9 in an amine-based buffer. Our optical and mass spectrometric characterizations identify 8-formyl flavin early in the process and 8-amino flavins (8AFs) at later times. The latter have not previously been documented in an ETF to our knowledge. Mass spectrometry of flavin products formed in Tris or bis-tris-aminopropane solutions demonstrates that the source of the amine adduct is the buffer. Stepwise reduction of the 8AF demonstrates that it can explain a charge transfer band observed near 726 nm in Bf ETF, as a complex involving the hydroquinone state of the 8AF in the ET site with the oxidized state of unmodified flavin in the Bf site. This supports the possibility that Bf ETF can populate a conformation enabling direct electron transfer between its two flavins, as has been proposed for cofactors brought together in complexes between ETF and its partner proteins.
|Journal||Journal of Biological Chemistry|
|State||Published - Dec 2022|
Bibliographical noteFunding Information:
N. M. R. and A.-F. M. acknowledge partial support from N.S.F. CHE 1808433 and CHE 2108134, and A.-F. M. additionally acknowledges partial support from D.O.E. DE-SC0021283 and the Einstein Foundation of Berlin for a visiting fellowship. C. V. G., and R. J. S. gratefully acknowledge partial support from the National Atmospheric and Space Administration’s Exobiology program (80NSSC17K0033).
© 2022 The Authors
- Charge-transfer complex
- Conformation change
- Electron bifurcation
- Electron transferring flavoprotein
- Modified flavin
ASJC Scopus subject areas
- Molecular Biology
- Cell Biology