Distinct Roles of ADP and Magnesium in the Structural Remodeling and Higher-Order Assembly of Peroxiredoxin 2

2-Cys peroxiredoxins (Prxs) undergo oligomeric transitions linked to peroxidase and chaperone activities, but the contributions of nucleotides and cations to lipid-dependent assembly remain unclear. We examined phosphatidylserine (PS)-induced Prx2 oligomers using biochemical assays and high-speed atomic force microscopy (HS-AFM). Both wild-type (WT) and P44A/S76A mutant (MT) Prx2 formed chaperone-active oligomers, although MT assembled more slowly and exhibited a more compact lateral geometry. Mg disassembled both oligomers, whereas ADP alone predominantly disassembled WT. Under combined Mg-ADP conditions, HS-AFM directly captured oligomer disassembly in both proteins, and separate experiments demonstrated higher-molecular-weight (HMW) assembly. WT HMW assemblies exhibited greater heights and stronger Mant-ADP co-elution than MT. Structural predictions suggested altered local Ser76-mediated contacts without major backbone changes. These results distinguish PS-induced functional switching from Mg- and ADP-dependent remodeling and support, without directly proving, a disassembly-reassembly route to HMW formation.