Outer sphere mutagenesis of Lactobacillus plantarum manganese catalase disrupts the cluster core

Mechanistic implications

Mei M. Whittaker, Vladimir V. Barynin, Takao Igarashi, James Whittaker

Research output: Contribution to journalArticle

35 Citations (Scopus)

Abstract

X-ray crystallography of the nonheme manganese catalase from Lactobacillus plantarum (LPC) [Barynin, V.V., Whittaker, M.M., Antonyuk, S.V., Lamzin, V.S., Harrison, P.M., Artymiuk, P.J. & Whittaker, J.W. (2001) Structure 9, 725-738] has revealed the structure of the dimanganese redox cluster together with its protein environment. The oxidized [Mn(III)Mn(III)] cluster is bridged by two solvent molecules (oxo and hydroxo, respectively) together with a μ1,3 bridging glutamate carboxylate and is embedded in a web of hydrogen bonds involving an outer sphere tyrosine residue (Tyr42). A novel homologous expression system has been developed for production of active recombinant LPC and Tyr42 has been replaced by phenylalanine using site-directed mutagenesis. Spectroscopic and structural studies indicate that disruption of the hydrogen-bonded web significantly perturbs the active site in Y42F LPC, breaking one of the solvent bridges and generating an 'open' form of the dimanganese cluster. Two of the metal ligands adopt alternate conformations in the crystal structure, both conformers having a broken solvent bridge in the dimanganese core. The oxidized Y42F LPC exhibits strong optical absorption characteristic of high spin Mn(III) in low symmetry and lower coordination number. MCD and EPR measurements provide complementary information defining a ferromagnetically coupled electronic ground state for a cluster containing a single solvent bridge, in contrast to the diamagnetic ground state found for the native cluster containing a pair of solvent bridges. Y42F LPC has less than 5% of the catalase activity and much higher Km, for H2O2 (≈1.4 M) at neutral pH than WT LPC, although the activity is slightly restored at high pH where the cluster is converted to a diamagnetic form. These studies provide new insight into the contribution of the outer sphere tyrosine to the stability of the dimanganese cluster and the role of the solvent bridges in catalysis by dimanganese catalases.

Original languageEnglish (US)
Pages (from-to)1102-1116
Number of pages15
JournalEuropean Journal of Biochemistry
Volume270
Issue number6
DOIs
StatePublished - Mar 2003

Fingerprint

Lactobacillus plantarum
Mutagenesis
Catalase
Ground state
Tyrosine
Hydrogen
X ray crystallography
X Ray Crystallography
Site-Directed Mutagenesis
Catalysis
Phenylalanine
Light absorption
Oxidation-Reduction
Paramagnetic resonance
Conformations
Glutamic Acid
Catalytic Domain
Hydrogen bonds
Crystal structure
Metals

Keywords

  • Catalase
  • Manganese
  • Redox
  • Spectroscopy
  • X-ray structure

ASJC Scopus subject areas

  • Biochemistry

Cite this

Outer sphere mutagenesis of Lactobacillus plantarum manganese catalase disrupts the cluster core : Mechanistic implications. / Whittaker, Mei M.; Barynin, Vladimir V.; Igarashi, Takao; Whittaker, James.

In: European Journal of Biochemistry, Vol. 270, No. 6, 03.2003, p. 1102-1116.

Research output: Contribution to journalArticle

Whittaker, Mei M. ; Barynin, Vladimir V. ; Igarashi, Takao ; Whittaker, James. / Outer sphere mutagenesis of Lactobacillus plantarum manganese catalase disrupts the cluster core : Mechanistic implications. In: European Journal of Biochemistry. 2003 ; Vol. 270, No. 6. pp. 1102-1116.
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AU - Whittaker, James

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AB - X-ray crystallography of the nonheme manganese catalase from Lactobacillus plantarum (LPC) [Barynin, V.V., Whittaker, M.M., Antonyuk, S.V., Lamzin, V.S., Harrison, P.M., Artymiuk, P.J. & Whittaker, J.W. (2001) Structure 9, 725-738] has revealed the structure of the dimanganese redox cluster together with its protein environment. The oxidized [Mn(III)Mn(III)] cluster is bridged by two solvent molecules (oxo and hydroxo, respectively) together with a μ1,3 bridging glutamate carboxylate and is embedded in a web of hydrogen bonds involving an outer sphere tyrosine residue (Tyr42). A novel homologous expression system has been developed for production of active recombinant LPC and Tyr42 has been replaced by phenylalanine using site-directed mutagenesis. Spectroscopic and structural studies indicate that disruption of the hydrogen-bonded web significantly perturbs the active site in Y42F LPC, breaking one of the solvent bridges and generating an 'open' form of the dimanganese cluster. Two of the metal ligands adopt alternate conformations in the crystal structure, both conformers having a broken solvent bridge in the dimanganese core. The oxidized Y42F LPC exhibits strong optical absorption characteristic of high spin Mn(III) in low symmetry and lower coordination number. MCD and EPR measurements provide complementary information defining a ferromagnetically coupled electronic ground state for a cluster containing a single solvent bridge, in contrast to the diamagnetic ground state found for the native cluster containing a pair of solvent bridges. Y42F LPC has less than 5% of the catalase activity and much higher Km, for H2O2 (≈1.4 M) at neutral pH than WT LPC, although the activity is slightly restored at high pH where the cluster is converted to a diamagnetic form. These studies provide new insight into the contribution of the outer sphere tyrosine to the stability of the dimanganese cluster and the role of the solvent bridges in catalysis by dimanganese catalases.

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