1QNM

HUMAN MANGANESE SUPEROXIDE DISMUTASE MUTANT Q143N

Summary for 1QNM

• Entry DOI: 10.2210/pdb1qnm/pdb
• Descriptor: MANGANESE SUPEROXIDE DISMUTASE, MANGANESE (II) ION (3 entities in total)
• Functional Keywords: oxidoreductase, manganese superoxide dismutase, hmnsod
• Biological source: Homo sapiens (human)
• Cellular location: Mitochondrion matrix: P04179
• Total number of polymer chains: 2
• Total formula weight: 44548.04

Authors

Guan, Y.,Tainer, J.A. (deposition date: 1997-07-03, release date: 1998-01-07, Last modification date: 2024-05-22)

Primary citation

Hsieh, Y.,Guan, Y.,Tu, C.,Bratt, P.J.,Angerhofer, A.,Lepock, J.R.,Hickey, M.J.,Tainer, J.A.,Nick, H.S.,Silverman, D.N.
Probing the active site of human manganese superoxide dismutase: the role of glutamine 143.
Biochemistry, 37:4731-4739, 1998

PubMed 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.

PubMed: 9537988
DOI: 10.1021/bi972395d

Experimental method

X-RAY DIFFRACTION (2.3 Å)