Insight into the high-potential branch of the alternate NAD+-dependent NADPH:ferredoxin oxidoreductase II (NfnII) from Pyrococcus furiosus: evidence for the gating step in electron bifurcation
Year: 2025
Type: article
Abstract: We have investigated the rapid-reaction kinetics of the NAD+-dependent NADPH:ferredoxin oxidoreductase II (NfnII) from Pyrococcus furiosus, permitting comparison with work done with the paralog NAD+-dependent NADPH:ferredoxin oxidoreductase (NfnI). The half-potentials of the electron-bifurcating L-FAD are highly crossed in NfnI, meaning that the potential for the quinone/semiquinone couple is significantly lower than that for the semiquinone/hydroquinone, so that the semiquinone oxidation state is thermodynamically unstable. The same appears to be true with NfnII, based on similar behavior in transient absorption spectroscopy experiments and the absence of FAD•- accumulation in the course of reductive titrations (manifested as a transient increase in absorbance at ∼380 nm). Reductive titrations with both the 1-electron donor sodium dithionite and two-electron donor NADPH demonstrate that, unlike NfnI, little FADH• accumulates in the high-potential pathway of NfnII, reflected in the absence of a transient increase in absorbance in the 500-600 nm region. This indicates that the half-potentials of the S-FAD are crossed in NfnII by a minimum of 120 mV. Rapid-reaction experiments mixing oxidized NfnII with NADPH also show no evidence of S-FADH• accumulation. Furthermore, the enzyme is partially reduced at the end of the reaction with NADPH, indicating that there is little electron transfer into the high-potential pathway of NfnII. When the reaction is done in the presence of ferredoxin, only one equivalent of ferredoxin becomes reduced, in contrast to the maximum of three equivalents seen with NfnI. This is consistent with only a single electron-bifurcation event occurring under these conditions with NfnII, with only a single high-potential electron passing into the [2Fe-2S] cluster of the high-potential pathway but does not progress further to the S-FAD. The lack of electron transfer into the S-FAD of NfnII due to its crossed half-potentials prevents successive bifurcation activity and indicates that electron transfer within the high-potential branch gates bifurcation activity in NfnI.
Source: Journal of Biological Chemistry
Language: English
Field: Energy
Domain: Physical Sciences
Open Access status: gold
Estimated APC paid: $3,430
Awards: G14908310, G1723423531, STRUCTURE, FUNCTION AND REACTIVITY OF CO DEHYDROGENASE FROM OLIGOTROPHA CARBOXIDOVORANS, G4704849243, G566293355, G5873644063, G6796042841, G7021897321, G7626931543, G847654844, HYPOTHERMOPHILIC MULTIPROTEIN COMPLEXES AND PATHWAYS FOR ENERGY CONSERVATION AND CATALYSIS, G924803583, G944719100
OpenAlex ID: W4413365334