EPR spectroscopy and catalase activity of manganese-bound DNA-binding protein from nutrient starved cells

Abstract

DNA-binding proteins from nutrient-starved cells (DPS) protect cells from oxidative stress by removing H₂O₂ and iron. A new class of DPS-like proteins has recently been identified, with DPS-like protein from Sulfolobus solfataricus (SsDPS) being the best characterized to date. SsDPS protects cells from oxidative stress and is upregulated in response to H₂O₂ but also in response to iron depletion. The ferroxidase active site of SsDPS is structurally similar to the active sites of manganese catalase and rat liver arginase. The present work shows that the ferroxidase center in SsDPS binds two Mn²⁺ ions with K _D = (1/K ₁ K ₂)^1/2 = 48(3) μM. The binding constant of the second Mn²⁺ is significantly higher than that of the first, inducing dinuclear Mn(II) cluster formation for all but the lowest concentrations of added Mn²⁺. In competition experiments, equimolar amounts of Fe²⁺ were unable to displace the bound manganese. EPR spectroscopy of the Mn₂ ²⁺ cluster showed signals comparable to those of other characterized dimanganese clusters. The exchange coupling for the cluster was determined, J = −1.4(3) cm⁻¹ (H = −2JS ₁ S ₂), and is within the range expected for a μ_1,1-carboxylato bridge between the manganese ions. Manganese-bound SsDPS showed catalase activity at a rate 10–100 times slower than for manganese catalases. EPR spectra of SsDPS after addition of H₂O₂ showed the appearance of an intermediate in the reaction with H₂O₂.