Probing the Active Site of Human Manganese Superoxide Dismutase:  The Role of Glutamine 143^,

Abstract

Structural and biochemical characterization of the nonliganding residue glutamine 143 near the manganese of human Mn superoxide dismutase (hMnSOD), a homotetramer of 22 kDa, reveals a functional role for this residue. In the wild-type protein, the side-chain amide group of Gln 143 is about 5 A from the metal and is hydrogen-bonded to Tyr 34, which is a second prominent side chain adjacent to the metal. We have prepared the site-specific mutant of hMnSOD with the conservative replacement of Gln 143 --> Asn (Q143N). The crystal structure of Q143N shows that the side-chain amide nitrogen of residue 143 is 1.7 A more distant from the manganese than in the wild-type enzyme. The Tyr 34 side-chain hydroxyl in Q143N is also moved to become 0.6 A more distant from the metal due to an additional water molecule. Differential scanning calorimetry showed that Q143N is slightly more stable than the wild-type enzyme with Tm for the main unfolding transition increased by 2 degrees C to 90.7 degrees C. Pulse radiolysis and stopped-flow spectrophotometry reveal that unlike wild-type hMnSOD, which is strongly inhibited by peroxide, Q143N MnSOD exhibits no product inhibition even at concentrations of O2. - in the millimolar range, and its catalysis follows Michaelis kinetics with no evidence of cooperativity. However, the overall catalytic activity of this mutant was decreased 2-3 orders of magnitude compared with the wild-type MnSOD, which can account for its lack of product inhibition. Q143N MnSOD lacked the visible absorption spectrum typical of wild-type Mn(III)SOD. Also, unlike the wild-type Mn(III)SOD, which is electron paramagnetic resonance (EPR) silent, Q143N MnSOD has a complex EPR spectrum with many resonances in the region below 2250 G. We conclude that the Gln 143 --> Asn mutation has increased the reduction potential of manganese to stabilize Mn(II), indicating that Gln 143 has a substantial role in maintaining a reduction potential favorable for the oxidation and reduction cycles in the catalytic disproportionation of superoxide. A solvent hydrogen isotope effect near 2 for kcat in catalysis by Q143N hMnSOD indicates rate-contributing proton transfers to form product hydroperoxide anion or hydrogen peroxide. The data demonstrate a prominent role for Gln 143 in maintaining the microenvironment of the manganese and in efficient catalysis of superoxide dismutation to oxygen and hydrogen peroxide.

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

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Novel Insights into the Basis for Escherichia coli Superoxide Dismutase's Metal Ion Specificity from Mn-Substituted FeSOD and Its Very High Em

“…A number of recent studies on metal-substituted SODs have implicated specific amino acid residues in their puzzling inactivity (26)(27)(28)48) and proposed thermodynamic bases for it (29,31). However, most of these studies have focused on Fe(Mn)SOD.…”

“…Mutation of Gln146 of MnSOD dramatically affects activity (48,52). Similarly, repositioning of the Gln of MnSOD to the position characteristic of FeSOD confers Fesupported activity on MnSOD (26), and movement of the Gln characteristic of FeSOD to the position characteristic of MnSOD increases Mn-supported activity in P. gingiValis SOD (27).…”

Plasma Antioxidant Activity as a Marker for a Favourable Outcome in Acute Ischemic Stroke

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