Contrasting roles for two conserved arginines: Stabilizing flavin semiquinone or quaternary structure, in bifurcating electron transfer flavoproteins

Nishya Mohamed-Raseek, Anne Frances Miller

Research output: Contribution to journalArticlepeer-review

4 Scopus citations


Bifurcating electron transfer flavoproteins (Bf ETFs) are important redox enzymes that contain two flavin adenine dinucleotide (FAD) cofactors, with contrasting reactivities and complementary roles in electron bifurcation. However, for both the "electron transfer" (ET) and the "bifurcating" (Bf) FADs, the only charged amino acid within 5 Å of the flavin is a conserved arginine (Arg) residue. To understand how the two sites produce different reactivities utilizing the same residue, we investigated the consequences of replacing each of the Arg residues with lysine, glutamine, histidine, or alanine. We show that absence of a positive charge in the ET site diminishes accumulation of the anionic semiquinone (ASQ) that enables the ET flavin to act as a single electron carrier, due to depression of the oxidized versus. ASQ reduction midpoint potential, E_ OX/ASQ. Perturbation of the ET site also affected the remote Bf site, whereas abrogation of Bf FAD binding accelerated chemical modification of the ET flavin. In the Bf site, removal of the positive charge impaired binding of FAD or AMP, resulting in unstable protein. Based on pH dependence, we propose that the Bf site Arg interacts with the phosphate(s) of Bf FAD or AMP, bridging the domain interface via a conserved peptide loop ("zipper") and favoring nucleotide binding. We further propose a model that rationalizes conservation of the Bf site Arg even in non-Bf ETFs, as well as AMP's stabilizing role in the latter, and provides a mechanism for coupling Bf flavin redox changes to domain-scale motion.

Original languageEnglish
Article number101733
JournalJournal of Biological Chemistry
Issue number4
StatePublished - Apr 1 2022

Bibliographical note

Funding Information:
Acknowledgments—The authors are grateful to the National Science Foundation for support under awards numbers CLP-1808433 and CLP-2108134. The authors thank H. D. Duan for providing R165Kand R165H-RpaETF constructs, T. Creamer for access to the CD spectrapolarimeter, A. Sebesta and B. Gess for maintenance of vital research infrastructure, S. Wang and A. Iyer for access to mass spectrometry, K. Ranguelova for guidance with EPR, and M. Ilham.

Publisher Copyright:
© 2022 American Society for Biochemistry and Molecular Biology Inc.. All rights reserved.

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology


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