Calorimetric Studies on the Tight Binding Metal Interactions of Escherichia coli Manganese Superoxide Dismutase

Abstract

Escherichia coli apomanganese superoxide dismutase, prepared by removing the native metal ion under denaturing conditions, exhibits thermally triggered metal uptake behavior previously observed for thermophilic and hyperthermophilic superoxide dismutases but over a lower temperature range. Superoxide dismutases (SODs) 1 (E.C. 1.15.1.1) are important antioxidant metalloenzymes protecting cells against oxidative stress arising from reactive oxygen species (1-4). SODs are ubiquitous, and multiple forms often exist within a single organism or cell. Four major forms of SODs (Mn, Fe, Cu/Zn, and Ni) have been identified, distinguished by the protein fold and by the nature of the catalytic metal ion (5-8). Mn-and Fe-SODs share extensive homology in protein structure, whereas the Ni and Cu/Zn enzymes are structurally distinct. Despite their similar structures (9), the Mn-and Fe-SODs exhibit a strict specificity for the catalytic metal ion (manganese or iron). Much lower selectivity is generally observed for metal binding, and the dimeric Mn-SOD from Escherichia coli is typically isolated as a mixture of Mn 2 -, (Mn,Fe)-, Fe 2 -, and half-apoforms (10, 11). Both the manganese-and iron-replete forms may be enriched by supplementing the culture medium with either manganese or iron salts (12). Apo-Mn-SOD binds metals nonselectively in the presence of guanidinium denaturants, and this method has been used to prepare manganese-, iron-, and cobalt-reconstituted forms (13-17).Each subunit of the E. coli Mn-SOD homodimer is composed of two domains, a predominantly ␣-helical N-terminal domain and a mixed ␣/␤ C-terminal domain (18). The metal-binding site lies on the interface between these two domains buried in the interior of the protein. The mononuclear metal ion is coordinated by four amino acid side chains (Fig. 1), two arising from the N-terminal domain (His-26 and His-81) and two from the C-terminal domain (Asp-167 and His-171), resulting in a metal ion cross-link between the two domains. A buried solvent molecule serves as a fifth ligand forming a hydrogen bond in the outer sphere of the metal complex with the amido head group of Glu-146 (E. coli Mn-SOD sequence numbering). The metal ion appears to be very tightly bound, being released only under denaturing conditions and resisting extraction by metal chelators like EDTA (13).Earlier work has shown that metal-free apo-SOD is produced during expression of recombinant thermophilic SODs in the mesophilic host, E. coli. The purified apoprotein has been shown to efficiently take up metal ion in vitro at elevated temperatures, and the binding is essentially irreversible under these conditions. At lower temperatures, metal uptake is not detected (19,20). The sigmoidal temperature profile for metalation of the recombinant thermophilic apo-SODs suggests that a two-state activated process is involved. The requirements for denaturing conditions to remove the metal and thermally triggered metal uptake imply the existence of large activation barriers for changes in the metalation state ...

Manganese Binds to Clostridium difficile Fbp68 and Is Essential for Fibronectin Binding

“…For example, when manganese binds to manganese superoxide dismutase of E. coli, the protein structure is stabilized as evidenced by the increased T m of manganese-bound proteins in thermo-unfolding experiments (apo-superoxide dismutase, T m ϭ 52.5°C; manganese superoxide dismutase, T m ϭ 68.6°C) (44). Likewise, manganese-bound prion protein resists higher concentrations of protease K, which also indicates the protein structure is stabilized by manganese (apo-prion, protease K ϭ 2 g/ml; manganese prion, protease K Ͼ25 g/ ml) (43).…”

“…To acquire environmental manganese, the binding affinity of most manganese-binding proteins must be high (e.g. E. coli manganese superoxide dismutase, K D ϭ 3.12 nM) (44). Thus, the high affinity of Fbp68 for manganese would help overcome the low concentration of manganese both in vivo and in the environment.…”

“…2ϩ binding is generally recognized as maintaining protein stability (43,44). To gain more insight about the function of manganese binding to Fbp68, DSC thermounfolding and thermo-denatured CD were performed.…”

Iron-Mediated Inhibition of Mitochondrial Manganese Uptake Mediates Mitochondrial Dysfunction in a Mouse Model of Hemochromatosis

“…In Saccharomyces cerevisiae, conditions of mitochondrial iron overload, or manganese deficiency, result in the mismetallation with iron of the MnSOD homologue, SOD2 (48). Inappropriate iron binding to MnSOD inactivates the protein because of altered redox potential (48)(49)(50)(51)(52)(53). In this mouse model, however, there was no demonstrable misincorporation of iron into SOD2, even in iron-fed wild type mice that accumulate mitochondrial iron (data not shown).…”

Biophysical and biochemical characterization of a liposarcoma‐derived recombinant MnSOD protein acting as an anticancer agent

“…Therefore, this protein is less stable than the human native enzyme. For the latter, differential scanning calorimetry 22 showed the first inactivation step at 68°C, followed by a main unfolding transition at about 88°C. Metal analysis, performed by atomic absorption spectroscopy, revealed that about two thirds of the metal binding sites are occupied by manganese ions.…”