PROBING THE ACTIVE-SITE OF HUMAN MANGANESE SUPEROXIDE-DISMUTASE - THEROLE OF GLUTAMINE-143

Structural and biochemical characterization of the nonliganding residu e glutamine 143 near the manganese of human Mn superoxide dismutase (h MnSOD), a homotetramer of 22 kDa, reveals a functional role for this r esidue. In the wild-type protein, the side-chain amide group of Gin 14 3 is about 5 Angstrom 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 rep lacement of Gln 143 --> Asn (Q143N). The crystal structure of Q143N sh ows that the side-chain amide nitrogen of residue 143 is 1.7 Angstrom 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 Angstrom m ore distant from the metal due to an additional water molecule. Differ ential scanning calorimetry showed that Q143N is slightly more stable than the wild-type enzyme with T-m for the main unfolding transition i ncreased by 2 degrees C to 90.7 degrees C. Pulse radiolysis and stoppe d-flow spectrophotometry reveal that unlike wild-type hMnSOD, which is strongly inhibited by peroxide, Q143N MnSOD exhibits no product inhib ition even at concentrations of O-2(.-) in the millimolar range, and i ts catalysis follows Michaelis kinetics with no evidence of cooperativ ity. However, the overall catalytic activity of this mutant was decrea sed 2-3 orders of magnitude compared with the wild-type MnSOD, which c an account for its lack of product inhibition. Q143N MnSOD lacked the visible absorption spectrum typical of wild-type Mn(m)SOD. Also, unlik e the wild-type Mn(III)-SOD, which is electron paramagnetic resonance (EPR) silent, Q143N MnSOD has a complex EPR spectrum with many resonan ces in the region below 2250 G. We conclude that the Gin 143 --> Asn m utation has increased the reduction potential of manganese to stabiliz e Mn(II), indicating that Gln 143 has a substantial role in maintainin g a reduction potential favorable for the oxidation and reduction cycl es in the catalytic disproportionation of superoxide. A solvent hydrog en isotope effect near 2 for k(cat) in catalysis by Q143N hMnSOD indic ates rate-contributing proton transfers to form product hydroperoxide anion or hydrogen peroxide. The data demonstrate a prominent role for Gin 143 in maintaining the microenvironment of the manganese and in ef ficient catalysis of superoxide dismutation to oxygen and hydrogen per oxide.