Cryo-EM structures define the electron bifurcating flavobicluster and ferredoxin binding site in an archaeal Nfn-Bfu transhydrogenase.

Flavin-based electron bifurcation couples exergonic and endergonic redox reactions in one enzyme complex to circumvent thermodynamic barriers and minimize free energy loss. Two unrelated enzymes designated NfnSL and NfnABC catalyze the NADPH-dependent reduction of ferredoxin and NAD. Bifurcation by NfnSL resides with a single FAD but the bifurcation mechanism of NfnABC, which represents the diverse and ubiquitous Bfu enzyme family, is completely different and largely unknown. Using cryo-EM structures of an archaeal NfnABC, we show that its bifurcation site is a flavobicluster consisting of FMN, one [4Fe-4S] and one [2Fe-2S] cluster where zinc atoms replace two additional clusters previously identified in other Bfu enzymes. NADH binds to the flavobicluster site of NfnABC and induces conformational changes that allow ferredoxin to bind between the C-terminal domains of NfnC and NfnB. Site-directed mutational analyses support the proposed mechanism that is likely conserved in all members of the Bfu enzyme family.