Rapid-reaction kinetics of the bifurcating NAD+-dependent NADPH:ferredoxin oxidoreductase NfnI from Pyrococcus furiosus

Journal Article · Fri Dec 01 00:00:00 EST 2023 · Journal of Biological Chemistry

DOI:https://doi.org/10.1016/j.jbc.2023.105403· OSTI ID:2204300

Ortiz, Steve; Niks, Dimitri; Wiley, Seth ; Lubner, Carolyn E. ; Hille, Russ

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 k_red of 36 ± 1.6 s⁻¹ and apparent K_d^NADPH 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.

• Research Organization: National Renewable Energy Laboratory (NREL), Golden, CO (United States)
• Sponsoring Organization: USDOE Office of Science (SC), Basic Energy Sciences (BES)
• Grant/Contract Number: AC36-08GO28308; SC00010666
• OSTI ID: 2204300
• Alternate ID(s): OSTI ID: 2205500
• Report Number(s): NREL/JA-2700-86866; S0021925823024316; 105403; PII: S0021925823024316
• Journal Information: Journal of Biological Chemistry, Journal Name: Journal of Biological Chemistry Vol. 299 Journal Issue: 12; ISSN 0021-9258
• Publisher: Elsevier
• Country of Publication: United States
• Language: English

Related Subjects

59 BASIC BIOLOGICAL SCIENCES
37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY
electron bifurcation
electron paramagnetic resonance
electron transfer
flavoprotein
kinetics