The ternary complex of Mn, synthetic decapeptide DP1 (DEHGTAVMLK), and orthophosphate is a superb antioxidant.

Mn coordinated by orthophosphate (Pi), metabolites, or peptides acts as a superoxide dismutase (SOD), and these Mn antioxidant complexes are universally accumulated in extremely radiation-resistant cell types across the tree of life. This behavior prompted design of decapeptide DP1 (DEHGTAVMLK) as a Mn ligand, and development of a highly potent Mn-antioxidant (MDP) containing [Pi] = 25 mM, and [DP1] = 3 mM, the ratio found in the radioresistant bacterium , with [Mn] = 1 mM. MDP is an exceptional antioxidant, both in vitro and in vivo, and has reinvigorated the development of radiation-inactivated whole-cell vaccines. This study investigates the nature of the active Mn complex in MDP. We measure the affinity of DP1 for the substitutionally labile Mn ion using isothermal-titration calorimetry (ITC) and use changes in the Mn solution EPR spectrum to determine affinities of Mn for DP1 and for Pi, and to monitor Mn ligation while titrated with the fixed Pi/DP1 ratio of MDP, 25/3, using ENDOR/ESEEM to characterize DP1 ligation to Mn. In parallel, H NMR of DP1 was used to monitor binding interactions between Pi and DP1, and DP1 binding to the diamagnetic Ca. We report: i) DP1 forms an extremely weak, dynamic Mn complex (K ≈ 40 M) ii) Mn binds Pi much more strongly (K ≈ 390 M) as shown previously, but iii) DP1 and Pi jointly bind to Mn in MDP to form a ternary Mn (Pi) (DP1) complex with greater formation-constant than Pi alone (K ≈ 670 M). It is this ternary complex that is the superb antioxidant in MDP.