Transition metal activation reframes SAMHD1 regulation.

SAMHD1 is the lone human dNTP triphosphohydrolase and is intimately linked to HIV viral restriction, dNTP pool maintenance, resistance to chemotherapy, and the autoinflammatory Aicardi-Goutières syndrome. While its substrate promiscuity and nucleotide basis of activity have been extensively studied, the identity and mechanistic roles of its metal cofactors remain poorly defined. Here, we integrate elemental analysis, spectroscopy, protein cross-linking, and enzyme kinetics to elucidate the molecular mechanisms underlying metal-dependent activation and catalysis in SAMHD1. Our findings establish that transition metals are essential components of SAMHD1 function, highlight their overlooked role in allosteric regulation, and reveal a central role for iron in organizing the dinuclear active site. We show that iron is preferentially incorporated in one position of the bimetallic core, where it promotes recruitment of a second divalent metal ion required for activity. While manganese can substitute for iron, it alters the metal binding equilibria, highlighting the unique functional properties of iron. Notably, SAMHD1 exhibits metal cofactor promiscuity at the second metal site, accommodating diverse divalent metals with distinct effects on activity. Cumulatively, our findings establish iron as a core structural and functional determinant of SAMHD1 catalysis and reveal how transition metal selectivity and flexibility enable enzymatic activity across diverse cellular environments and metal flux conditions.