Lysine acetylation-mediated regulation of ferredoxin and ferredoxin reductase redox-active proteins in Haloferax volcanii.

Lysine acetylation is an evolutionarily conserved, post-translational modification that regulates metabolism and protein function, yet its role in archaeal electron transfer systems remains poorly understood. Here, we investigated lysine acetylation of the 2Fe-2S ferredoxin Hv Fdx (HVO_2995) and its flavin-dependent oxidoreductase Hv FdR (HVO_2345) partner in the halophilic archaeon Haloferax volcanii . Genetic and biochemical analyses established Hv Fdx as an essential 2Fe-2S ferredoxin with a midpoint redox potential of -385 mV. Lysine acetylation of Hv Fdx was found to occur primarily at K119, a residue positioned near the [Fe-S] cluster interface, and to modulate electron transfer capacity without impacting Fe-S cluster incorporation, midpoint potential, or protein abundance. In contrast, Hv FdR was found lysine acetylated at multiple sites in a manner consistent with a non-enzymatic mechanism that resulted in altered flavin binding, enzymatic activity, and thermal stability. Lysine acetylation of Hv Fdx was found to stimulate electron flow from Hv FdR as measured by an anaerobic NADPH → Hv FdR → Hv Fdx → DCIP assay. 3D structural modeling, proteomic, biochemical, and genetic assays suggest the haloarchaeal GNAT-family acetyltransferase homolog HVO_2874 as a candidate enzyme associated with Hv Fdx lysine acetylation and optimal growth of H. volcanii . Together, these findings demonstrate that lysine acetylation differentially regulates archaeal redox-active proteins and functions as an important mechanism coordinating redox metabolism in H. volcanii .