Site-Differentiated Iron-Sulfur Cluster Ligation Affects Flavin-Based Electron Bifurcation Activity

Courtney Wise, Anastasia Ledinina, Carolyn Lubner

Research output: Contribution to journalArticlepeer-review

2 Scopus Citations


Electron bifurcation is an elegant mechanism of biological energy conversion that effectively couples three different physiologically relevant substrates. As such, enzymes that perform this function often play critical roles in modulating cellular redox metabolism. One such enzyme is NADH-dependent reduced-ferredoxin: NADP+ oxidoreductase (NfnSL), which couples the thermodynamically favorable reduction of NAD+ to drive the unfavorable reduction of ferredoxin from NADPH. The interaction of NfnSL with its substrates is constrained to strict stoichiometric conditions, which ensures minimal energy losses from non-productive intramolecular electron transfer reactions. However, the determinants for this are not well understood. One curious feature of NfnSL is that both initial acceptors of bifurcated electrons are unique iron–sulfur (FeS) clusters containing one non-cysteinyl ligand each. The biochemical impact and mechanistic roles of site-differentiated FeS ligands are enigmatic, despite their incidence in many redox active enzymes. Herein, we describe the biochemical study of wild-type NfnSL and a variant in which one of the site-differentiated ligands has been replaced with a cysteine. Results of dye-based steady-state kinetics experiments, substrate-binding measurements, biochemical activity assays, and assessments of electron distribution across the enzyme indicate that this site-differentiated ligand in NfnSL plays a role in maintaining fidelity of the coordinated reactions performed by the two electron transfer pathways. Given the commonality of these cofactors, our findings have broad implications beyond electron bifurcation and mechanistic biochemistry and may inform on means of modulating the redox balance of the cell for targeted metabolic engineering approaches.

Original languageAmerican English
Article number823
Number of pages14
Issue number9
StatePublished - 2022

Bibliographical note

Publisher Copyright:
© 2022 by the authors.

NREL Publication Number

  • NREL/JA-2700-83672


  • biochemistry
  • bioenergetics
  • biological electron transfer
  • electron bifurcation
  • energy conservation
  • flavoenzyme
  • iron–sulfur cluster
  • metabolism
  • thermodynamics


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