Rapid-Reaction Kinetics of the Bifurcating NAD+-Dependent NADPH:Ferredoxin Oxidoreductase NfnI from Pyrococcus furiosus: Article No. 105403

Steve Ortiz, Dimitri Niks, Seth Wiley, Carolyn Lubner, Russ Hille

Research output: Contribution to journalArticlepeer-review


The rapid-reaction kinetic behavior of the NAD+-dependent NADPH:ferredoxin oxidoreductase (NfnI) has been investigated. NfnI is a heterodimeric transhydrogenase of the flavin-based electron bifurcating class of flavoproteins and in the course of catalysis takes two electron pairs from NADPH to reduce two ferredoxins and one NAD+ through two successive bifurcation events. NADPH reduction takes place at the bifurcating FAD of NfnI's large subunit, with high-potential electrons transferred to the [2Fe-2S] cluster and S-FADH of the small subunit, ultimately on to NAD+, and low-potential electrons transferred to two [4Fe-4S] clusters of the large subunit, ultimately on to ferredoxin. Reduction of NfnI by NADPH goes to completion only at higher pH, with a limiting kred of 36 +/- 1.6 s-1 and apparent KdNADPH of 5 +/- 1.2 uM. Reduction of one of the [4Fe-4S] clusters of NfnI occurs within a second, as monitored by electron paramagnetic resonance (EPR) spectroscopy, indicating that in the absence of NAD+ the system can bifurcate with electrons entering the low potential pathway. When enzyme is reduced by NADPH in the absence of NAD+ but the presence of ferredoxin, up to three equivalents of ferredoxin become reduced in the course of the reaction, although the reaction is considerably slower than steady-state turnover, likely due to a previously identified conformational change in NfnI. Bifurcation thus appears to be rate-limited by transfer of the first, high-potential electron into the high-potential pathway. The observation of ferredoxin reduction in the absence of NAD+ demonstrates that the process of electron bifurcation is an intrinsic property of the bifurcating FAD and not strictly dependent on the simultaneous presence of NAD+ and ferredoxin. The tight coupling between NAD+ and ferredoxin reduction observed in steady-state assays with NfnI is instead simply due to the need to remove reducing equivalents from the high-potential electron pathway under multiple-turnover conditions. Reoxidation of pre-reduced enzyme with NAD+ is well-behaved, with a limiting kox of 50 +/- 0.7 s-1 and apparent KdNAD+ of 8 +/- 0.6 uM.
Original languageAmerican English
Number of pages12
JournalJournal of Biological Chemistry
Issue number12
StatePublished - 2023

NREL Publication Number

  • NREL/JA-2700-86866


  • electron bifurcation
  • electron paramagnetic resonance
  • electron transfer
  • flavoprotein
  • kinetics


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