Abstract
Electron bifurcation has recently gained acceptance as the third mechanism of energy conservation in which energy is conserved through the coupling of exergonic and endergonic reactions. A structure-based mechanism of bifurcation has been elucidated recently for the flavin-based enzyme NADH-dependent ferredoxin NADP+ oxidoreductase I (NfnI) from the hyperthermophillic archaeon Pyrococcus furiosus. NfnI is thought to be involved in maintaining the cellular redox balance, producing NADPH for biosynthesis by recycling the two other primary redox carriers, NADH and ferredoxin. The P. furiosus genome encodes an NfnI paralog termed NfnII, and the two are differentially expressed, depending on the growth conditions. In this study, we show that deletion of the genes encoding either NfnI or NfnII affects the cellular concentrations of NAD(P)H and particularly NADPH. This results in a moderate to severe growth phenotype in deletion mutants, demonstrating a key role for each enzyme in maintaining redox homeostasis. Despite their similarity in primary sequence and cofactor content, crystallographic, kinetic, and mass spectrometry analyses reveal that there are fundamental structural differences between the two enzymes, and NfnII does not catalyze the NfnI bifurcating reaction. Instead, it exhibits non-bifurcating ferredoxin NADP oxidoreductase-type activity. NfnII is therefore proposed to be a bifunctional enzyme and also to catalyze a bifurcating reaction, although its third substrate, in addition to ferredoxin and NADP(H), is as yet unknown.
Highlights
Electron bifurcation has recently gained acceptance as the third mechanism of energy conservation in which energy is conserved through the coupling of exergonic and endergonic reactions
Functions of two Nfns in P. furiosus pools in some anaerobes are balanced by a bifurcating enzyme called NADH-dependent ferredoxin NADPϩ oxidoreductase (Nfn)
Genes encoding homologs of the S and L subunits of P. furiosus NADH-dependent ferredoxin NADP؉ oxidoreductase I (NfnI) were found in 72 archaeal genomes
Summary
Ferredoxin; Fdred, reduced ferredoxin; Fdox, oxidized ferredoxin; Nfn, NADH-dependent ferredoxin NADPϩ oxidoreductase; SH, soluble hydrogenase; FNOR, ferredoxin-dependent NAD(P)ϩ oxidoreductase; NSR, NADPH-dependent sulfur reductase; M, maltose; MS, maltose sulfur; PS, peptide sulfur; NadB, L-aspartate oxidase; BV, benzyl viologen; OE, overexpression; qPCR, quantitative PCR; RMSD, root mean square deviation; PDB, Protein Data Bank. Functions of two Nfns in P. furiosus pools in some anaerobes are balanced by a bifurcating enzyme called NADH-dependent ferredoxin NADPϩ oxidoreductase (Nfn). This was first reported in Clostridium kluyveri and catalyzes the production of NADPH coupled to the simultaneous oxidation of NADH and reduced Fd. This was first reported in Clostridium kluyveri and catalyzes the production of NADPH coupled to the simultaneous oxidation of NADH and reduced Fd This enzyme carried out flavin-based electron bifurcation, a fundamental mechanism of biological energy conservation [12, 13]. The second Nfn homolog of P. furiosus, termed NfnII (PF1910-11), has yet to be structurally and functionally characterized and is the subject of this study. We provide the first biochemical and structural characterization of P. furiosus NfnII
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