Probing the active site of human manganese superoxide dismutase: the role of glutamine 143.
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.
Hsieh
Y
Y
Department of Pharmacology and Therapeutics, University of Florida, Gainesville, Florida 32610, USA.
Guan
Y
Y
Tu
C
C
Bratt
P J
PJ
Angerhofer
A
A
Lepock
J R
JR
Hickey
M J
MJ
Tainer
J A
JA
Nick
H S
HS
Silverman
D N
DN
D001665:Binding Sites; D002384:Catalysis; D004578:Electron Spin Resonance Spectroscopy; D005973:Glutamine / Q000378:metabolism*; D006801:Humans; D006860:Hydrogen Bonding; D007700:Kinetics; D008969:Molecular Sequence Data; D013482:Superoxide Dismutase / Q000737:chemistry / Q000378:metabolism*; D013816:Thermodynamics
D005973:Glutamine; D013482:Superoxide Dismutase