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

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) mu M. 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 Mn-2 (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 mu(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.