Differential ligation alters electronic state and coupling signals of iron-sulfur clusters in flavin-based electron bifurcation.

Flavin-based electron bifurcation (FBEB) is employed by microorganisms for controlling pools of redox equivalents by reversibly splitting electron pairs into high- and low-energy levels from an initial midpoint potential. Our ability to harness this phenomenon is crucial for biocatalytic design which is limited by our understanding of energy coupling in the bifurcation system. In Pyrococcus furiosus, FBEB is carried out by the NADH-dependent ferredoxin:NADP-oxidoreductase (NfnSL), coupling the uphill reduction of ferredoxin in NfnL to the downhill reduction of NAD in NfnS from oxidation of NADPH.

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.

We have investigated the rapid-reaction kinetics of the NAD-dependent NADPH:ferredoxin oxidoreductase II (NfnII) from Pyrococcus furiosus, permitting a comparison with recent work done with the paralog NfnI from the same organism. The half-potentials of the electron-bifurcating L-FAD are highly crossed in both NfnI, meaning the potential for the quinone/semiquinone couple is significantly lower than that for the semiquinone/hydroquinone couple so that the semiquinone oxidation state is thermodynamically unstable. The same appears to be the case with NfnII on the basis of its similar behavior in transient absorption spectroscopy experiments and the absence of any evidence for FAD• accumulation in the course of reductive titrations (which would be manifested as a transient increase in absorbance at ∼380 nm).