A minimal SufB₂C₂ complex functions as a [4Fe-4S] cluster scaffold in methanogenic archaea

Iron-sulfur clusters are essential cofactors in all domains of life, yet their biogenesis in obligately anaerobic archaea remains poorly understood. Here, we characterized the minimal two-protein SUF system in methanogenic archaea, composed solely of SufB and SufC. Using Methanococcus maripaludis as a model, we demonstrate that the SUF proteins from its native host form a stable SufB₂C₂ heterotetramer that binds a [4Fe-4S] cluster via three conserved cysteines in SufC. Mutations of conserved cysteine and histidine residues of SufB do not impair cluster binding. The complex interacts with the SAM-containing methanogenesis marker protein 10 (MmpX), suggesting direct Fe-S cluster transfer from SufB₂C₂ to target proteins. Mutational analysis of Methanothermococcus thermolithotrophicus proteins confirmed that SufC is the primary cluster-binding component, while SufB enhances ATPase and cluster transfer activities. Evolutionary comparisons suggest that this two-protein SUF system represents an ancestral form of Fe-S cluster biogenesis.IMPORTANCEFe-S clusters are ancient and indispensable cofactors, yet their biosynthesis in obligately anaerobic archaea remains underexplored. This study provides mechanistic and evolutionary insights into the Fe-S cluster assembly machinery in methanogenic archaea. Unlike the bacterial six-component SUF systems, this minimal two-component SUF system (SufB₂C₂) operates without auxiliary proteins. Our findings expand the known diversity of Fe-S cluster biogenesis machineries and shed light on a potential evolutionary precursor adapted to the Earth's ancient anoxic environments. It also provides a foundation for engineering minimal Fe-S cluster biosynthesis pathways in synthetic biology applications.