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 H(2)O(2) 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 H(2)O(2) 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 Mn(2+) ions with K (D) = (1/K (1) K (2))(1/2) = 48(3) microM. The binding constant of the second Mn(2+) is significantly higher than that of the first, inducing dinuclear Mn(II) cluster formation for all but the lowest concentrations of added Mn(2+). In competition experiments, equimolar amounts of Fe(2+) 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 H(2)O(2) showed the appearance of an intermediate in the reaction with H(2)O(2).

Initial Steps of Photosystem II de Novo Assembly and Preloading with Manganese Take Place in Biogenesis Centers in Synechocystis

“…Based on titration and competition experiments with Ca 2+ and Mg 2+ ions, we postulate a stoichiometry of 1 Mn 2+ :1 PratA at this site (Figures 3 and 4). The observed K d1 value is in the range of affinities of other Mn 2+ binding proteins, such as the Mn-dependent catalase MnCat (K d = 40 mM) or the oxidative stress protecting protein SsDPS (K d = 48 mM) (Meier et al, 1996;Crowley et al, 2000;Pierce et al, 2003;Hayden and Hendrich, 2010). Nevertheless, much higher binding constants for Mn 2+ to protein have been reported, for instance, in case of MnSOD and PsbP proteins (Mizuno et al, 2004;Bondarava et al, 2007).…”

“…In the presence of rPratA, a significantly smaller EPR signal amplitude was observed in comparison to a rPratA-free sample containing identical Mn 2+ concentration in the same buffer ( Figure 3A). The reduction of the signal amplitude of the Mn 2+ spectrum suggests decreased amounts of free Mn 2+ in solution due to Mn 2+ binding to the protein (Reed and Cohn, 1970;Reed and Markham, 1984;Sen et al, 2006;Hayden and Hendrich, 2010). This effect was abolished upon denaturation of rPratA by 8M urea, indicating specific binding of Mn 2+ , as folding of the protein is crucial for Mn 2+ interaction ( Figure 3B).…”

The archaeal Dps nanocage targets kidney proximal tubules via glomerular filtration

“…solfataricus Dps exhibits a catalase-like activity when Mn is bound to an active site of the protein subunits (2 Mn per subunit) (11,25). Mn loading to Dps was performed following an established protocol (25). A stock solution of Mn (0.4 M) was prepared by dissolving MnCl 2 •4H 2 O in water.…”

“…In contrast, MnDps exerts antioxidant effects similar to those of Mn catalase. The added Mn is tightly bound to the ferroxidase center and not displaced by equimolar amounts of iron (25). Therefore, MnDps could provide stable and powerful antioxidant effects irrespective of free iron availability in vivo.…”

Iron-Sulfur World in Aerobic and Hyperthermoacidophilic ArchaeaSulfolobus

“…The stunning capability of some aerobic and thermoacidophilic archaea to grow at extremely low pH [19–22] has therefore implicit meaning in that the intracellular Fe-S world must be protected not only by scavenging reactive oxygen species (e.g., see [62–67]) but also by balancing the intracellular pH at an acceptable value (typically 5.6–6.5 in Sulfolobus and Thermoplasma [21, 58–60]) in the face of a huge proton gradient (∆pH = pH in − pH out ). The ∆pH across the cytoplasmic membrane of these archaea is intrinsically linked to the cellular energetics [21, 58, 68], because it is the primary contributor to the proton motive force (PMF)…”