Rapid-reaction kinetics of the bifurcating NAD+-dependent NADPH:ferredoxin oxidoreductase NfnI from Pyrococcus furiosus
Abstract
We have investigated the kinetics of NAD+-dependent NADPH:ferredoxin oxidoreductase (NfnI), a bifurcating transhydrogenase that takes two electron pairs from NADPH to reduce two ferredoxins and one NAD+ through 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+; low-potential electrons are transferred to two [4Fe-4S] clusters of the large subunit and 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 μM. Reduction of one of the [4Fe-4S] clusters of NfnI occurs within a second, indicating that in the absence of NAD+, the system can bifurcate and generate low-potential electrons without NAD+. When enzyme is reduced by NADPH in the absence of NAD+ but the presence of ferredoxin, up to three equivalents of ferredoxin become reduced, although the reaction is considerably slower than seen during steady-state turnover. Bifurcation appears to be limited by transfer of the first, high-potential electron into the high-potential pathway. Ferredoxin reduction without NAD+ demonstrates that electron bifurcation is an intrinsic property ofmore »
- Publication Date:
- Research Org.:
- National Renewable Energy Laboratory (NREL), Golden, CO (United States)
- Sponsoring Org.:
- USDOE Office of Science (SC), Basic Energy Sciences (BES)
- OSTI Identifier:
- 2204300
- Alternate Identifier(s):
- OSTI ID: 2205500
- Report Number(s):
- NREL/JA-2700-86866
Journal ID: ISSN 0021-9258; S0021925823024316; 105403; PII: S0021925823024316
- Grant/Contract Number:
- AC36-08GO28308; SC00010666
- Resource Type:
- Published Article
- Journal Name:
- Journal of Biological Chemistry
- Additional Journal Information:
- Journal Name: Journal of Biological Chemistry Journal Volume: 299 Journal Issue: 12; Journal ID: ISSN 0021-9258
- Publisher:
- Elsevier
- Country of Publication:
- United States
- Language:
- English
- Subject:
- 59 BASIC BIOLOGICAL SCIENCES; 37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY; electron bifurcation; electron paramagnetic resonance; electron transfer; flavoprotein; kinetics
Citation Formats
Ortiz, Steve, Niks, Dimitri, Wiley, Seth, Lubner, Carolyn E., and Hille, Russ. Rapid-reaction kinetics of the bifurcating NAD+-dependent NADPH:ferredoxin oxidoreductase NfnI from Pyrococcus furiosus. United States: N. p., 2023.
Web. doi:10.1016/j.jbc.2023.105403.
Ortiz, Steve, Niks, Dimitri, Wiley, Seth, Lubner, Carolyn E., & Hille, Russ. Rapid-reaction kinetics of the bifurcating NAD+-dependent NADPH:ferredoxin oxidoreductase NfnI from Pyrococcus furiosus. United States. https://doi.org/10.1016/j.jbc.2023.105403
Ortiz, Steve, Niks, Dimitri, Wiley, Seth, Lubner, Carolyn E., and Hille, Russ. Fri .
"Rapid-reaction kinetics of the bifurcating NAD+-dependent NADPH:ferredoxin oxidoreductase NfnI from Pyrococcus furiosus". United States. https://doi.org/10.1016/j.jbc.2023.105403.
@article{osti_2204300,
title = {Rapid-reaction kinetics of the bifurcating NAD+-dependent NADPH:ferredoxin oxidoreductase NfnI from Pyrococcus furiosus},
author = {Ortiz, Steve and Niks, Dimitri and Wiley, Seth and Lubner, Carolyn E. and Hille, Russ},
abstractNote = {We have investigated the kinetics of NAD+-dependent NADPH:ferredoxin oxidoreductase (NfnI), a bifurcating transhydrogenase that takes two electron pairs from NADPH to reduce two ferredoxins and one NAD+ through 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+; low-potential electrons are transferred to two [4Fe-4S] clusters of the large subunit and 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 μM. Reduction of one of the [4Fe-4S] clusters of NfnI occurs within a second, indicating that in the absence of NAD+, the system can bifurcate and generate low-potential electrons without NAD+. When enzyme is reduced by NADPH in the absence of NAD+ but the presence of ferredoxin, up to three equivalents of ferredoxin become reduced, although the reaction is considerably slower than seen during steady-state turnover. Bifurcation appears to be limited by transfer of the first, high-potential electron into the high-potential pathway. Ferredoxin reduction without NAD+ demonstrates that electron bifurcation is an intrinsic property of the bifurcating FAD and is not dependent on the simultaneous presence of NAD+ and ferredoxin. The tight coupling between NAD+ and ferredoxin reduction observed under multiple-turnover conditions is instead simply due to the need to remove reducing equivalents from the high-potential electron pathway under multiple-turnover conditions.},
doi = {10.1016/j.jbc.2023.105403},
journal = {Journal of Biological Chemistry},
number = 12,
volume = 299,
place = {United States},
year = {Fri Dec 01 00:00:00 EST 2023},
month = {Fri Dec 01 00:00:00 EST 2023}
}
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