Mechanistic insights into redox activity and catalytic determinants of the haloarchaeal flavin-dependent oxidoreductase Hv FdR

Members of the FAD-dependent oxidoreductase family (IPR050260) play diverse and key roles in maintaining cellular redox balance, yet the functions of many distinct subgroups within this family remain unknown. Here, we define the biochemical and physiological functions of the Haloferax volcanii flavin-dependent oxidoreductase Hv FdR (HVO_2345; fdr ), a haloarchaeal member of a previously uncharacterized IPR050260 subgroup. Hv FdR binds FAD and catalyzes NAD(P)H oxidase, diaphorase and ferredoxin reductase activities, with a kinetic preference for NADPH over NADH and catalytic properties that are strongly influenced by oxygen availability. Under stoichiometric conditions, Hv FdR mediates reverse electron transfer to NADP⁺, suggesting that intracellular nicotinamide nucleotide pools regulate electron flow bidirectionally. Consistent with this reversibility, Hv FdR bound-FAD exhibits a low midpoint redox potential (−413 mV), supporting its capacity to function as an electron donor. Deletion of fdr impairs growth and elevates intracellular NADPH levels, consistent with a role for Hv FdR in maintaining NADP(H) homeostasis. Conserved residues K47 and Y323 are identified as determinants of Hv FdR electron transfer activity and may function as a regulatory gate that modulates electron flow while limiting excessive H ₂ O ₂ production under aerobic conditions. Together, these findings establish Hv FdR as an oxygen-responsive flavin-dependent oxidoreductase that contributes to cellular redox homeostasis and provides functional insight into a previously uncharacterized subgroup of the IPR050260 family.