Transition Metal Activation Reframes SAMHD1 Regulation.

SAMHD1 is the lone human dNTP triphosphohydrolase and is linked to antiviral defense, nucleotide pool homeostasis, chemotherapy resistance, and the autoinflammatory Aicardi-Goutières syndrome. Although its substrate specificity and nucleotide-dependent oligomerization have been extensively studied, the identity and mechanistic roles of its metal cofactors remain poorly understood. Here, we integrate selective metal enrichment, spectroscopy, biochemical reconstitution, and enzyme kinetics to define the metal requirements underlying SAMHD1 activation and catalysis. We show that robust SAMHD1 activity is preferentially supported by transition metals and that the enzyme readily assembles multiple iron-containing dinuclear active sites in solution. Iron preferentially binds to one position of the bimetallic core and promotes recruitment of a second divalent metal required for catalysis. Although manganese can substitute for iron, it alters metal-binding equilibria and less efficiently supports dinuclear cofactor assembly, highlighting a specialized organizational role for iron. In contrast, the second site remains comparatively permissive and accommodates various divalent metal ions with distinct functional consequences. Mixed-metal active sites further retain catalytic activity across redox conditions that otherwise suppress activity in homodinuclear diiron configurations, suggesting that metal plasticity buffers SAMHD1 against oxidative inhibition. Transition metals additionally act as higher-affinity allosteric activators than Mg2+, revealing that metal identity contributes to both catalytic and regulatory layers of SAMHD1 function. Cumulatively, these findings redefine the metal requirements of SAMHD1 and establish a framework in which iron-dependent active site organization and mixed-metal flexibility cooperate to sustain dNTP hydrolysis under changing cellular environments and metal flux conditions.