Mutagenesis of a proton linkage pathway in Escherichia coli manganese superoxide dismutase

Mei M. Whittaker, James Whittaker

Research output: Contribution to journalArticle

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Abstract

Mutagenesis of Escherichia coli manganese superoxide dismutase (MnSD) demonstrates involvement of the strictly conserved gateway tyrosine (Y34) in exogenous ligand interactions. Conservative replacement of this residue by phenylalanine (Y34F) affects the pH sensitivity of the active-site metal ion and perturbs ligand binding, stabilizing a temperature-independent six- coordinate azide complex. Mutant complexes characterized by optical and electron paramagnetic resonance (EPR) spectroscopy are distinct from the corresponding wild-type forms and the anion affinities are altered, consistent with modified basicity of the metal ligands. However, dismutase activity is only slightly reduced by mutagenesis, implying that tyrosine-34 is not essential for catalysis and may function indirectly as a proton donor for turnover, coupled to a protonation cycle of the metal ligands. In vivo substitution of Fe for Mn in the MnSD wild-type and mutant proteins leads to increased affinity for azide and altered active-site properties, shifting the pH-dependent transition of the active site from 9.7 (Mn) to 6.4 (Fe) for wt enzyme. This pH-coupled transition shifts once more to a higher effective pK(a) for Y34F Fe2-MnSD, allowing the mutant to be catalytically active well into the physiological pH range and decreasing the metal selectivity of the enzyme. Peroxide sensitivities of the Fe complexes are distinct for the wild- type and mutant proteins, indicating a role for Y34 in peroxide interactions. These results provide evidence for a conserved peroxide-protonation linkage pathway in superoxide dismutases, analogous to the proton relay chains of peroxidases, and suggests that the selectivity of Mn and Fe superoxide dismutases is determined by proton coupling with metal ligands.

Original languageEnglish (US)
Pages (from-to)8923-8931
Number of pages9
JournalBiochemistry
Volume36
Issue number29
DOIs
StatePublished - Jul 22 1997

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Mutagenesis
Escherichia coli
Superoxide Dismutase
Protons
Metals
Ligands
Peroxides
Catalytic Domain
Azides
Protonation
Mutant Proteins
Tyrosine
Peroxidases
Electron Spin Resonance Spectroscopy
Enzymes
Alkalinity
Catalysis
Phenylalanine
Anions
Metal ions

ASJC Scopus subject areas

  • Biochemistry

Cite this

Mutagenesis of a proton linkage pathway in Escherichia coli manganese superoxide dismutase. / Whittaker, Mei M.; Whittaker, James.

In: Biochemistry, Vol. 36, No. 29, 22.07.1997, p. 8923-8931.

Research output: Contribution to journalArticle

Whittaker, Mei M. ; Whittaker, James. / Mutagenesis of a proton linkage pathway in Escherichia coli manganese superoxide dismutase. In: Biochemistry. 1997 ; Vol. 36, No. 29. pp. 8923-8931.
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