Combined Experimental and Theoretical Study To Characterize Plutonyl–Calmodulin Interactions

Abstract Plutonium, a highly radiotoxic heavy metal used in civil and military applications, can form stable oxidized species in solution, plutonyl cations PuVIO22+ and PuVO2+. Despite their toxicological relevance, their interactions with biological ligands remain poorly understood. Here, we investigate plutonyl binding to CaMΔ, an engineered calmodulin variant with high affinity for UVIO22+. Upon addition of plutonium to CaMΔ solutions buffered with 2-(N-morpholino)ethane sulfonic acid (MES), a 1:1 CaMΔ-PuVIO22+ complex is formed, followed by progressive reduction to PuVO2+. This reduction is slower than in MES buffer alone, indicating that CaMΔ stabilizes PuVIO22+. To investigate this phenomenon, binding loop variants were designed in silico and their plutonyl coordination was screened by molecular dynamics simulations. Promising candidates were produced and experimentally evaluated in vitro. Several factors appear to contribute to PuVIO22+ stabilization, including thermodynamic parameters such as ΔG, the number of negative charges in the metal-binding loop, and structural features such as hydrogen-bonding interactions involving coordinating carboxylates. These results provide insights into the mechanisms governing plutonium stabilization and redox behavior in biological environments, with implications for nuclear toxicology and actinide bioinorganic chemistry.