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. Flanking the bifurcating flavin are two site-differentiated iron‑sulfur clusters; the nearest is a glutamate-ligated [4Fe—4S] cluster in NfnL. Recent biochemical experiments substituting the native glutamate with cysteine led to loss of coupling between the uphill and downhill pathways, in contrast to the tight thermodynamic coupling in the native system. To understand how this decoupling is biochemically manifested by the cysteine-substituted [4Fe—4S] in NfnL, we employed electron paramagnetic resonance (EPR) spectroscopy to identify changes in electronic architecture and square wave voltammetry (SWV) to probe thermodynamic shifts produced by the substitution. We observed notable g-value shifts in the EPR for the cysteine-substituted iron‑sulfur cluster in addition to significant downward shifts in the redox potential, as well as the disappearance of several low-field signals observed in the native NfnSL complex. These results suggest the site-differentiated glutamate residue facilitates higher spin states in the [4Fe4S] cluster to bridge energetic gaps in electron transfer to the bifurcating flavin in the native complex, preventing unwanted short-circuiting seen in the cysteine-substituted complex.