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Membrane-associated methane monooxygenase from Methylococcus capsulatus (Bath)

J A Zahn1, A A DiSpirito

  • 1Department of Microbiology, Iowa State University, Ames 50011, USA.

Journal of Bacteriology
|February 1, 1996
PubMed
Summary

Researchers purified the membrane-associated methane monooxygenase (pMMO) enzyme from Methylococcus capsulatus Bath. This active enzyme preparation, crucial for methane oxidation, contained specific polypeptides and metal cofactors, revealing insights into its structure and function.

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Area of Science:

  • Biochemistry
  • Enzymology
  • Microbial Metabolism

Background:

  • Methane monooxygenase (MMO) enzymes are critical for methane oxidation in microorganisms.
  • Membrane-associated MMO (pMMO) plays a key role in the methane cycle but its purification and characterization remain challenging.

Purpose of the Study:

  • To isolate and characterize an active preparation of pMMO from Methylococcus capsulatus Bath.
  • To identify the protein subunits and metal composition of the purified pMMO.
  • To investigate the functional properties and spectroscopic features of the purified pMMO.

Main Methods:

  • Purification using ion-exchange and hydrophobic interaction chromatography with dodecyl beta-D-maltoside detergent.
  • Analysis of polypeptide composition by molecular mass.

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  • Spectroscopic analysis including electron paramagnetic resonance (EPR).
  • Enzyme activity assays using propylene oxidation with duroquinol as reductant.

    • Main Results:

    • An active pMMO preparation was obtained, consisting of 47,000, 27,000, and 25,000 Da polypeptides.
    • The 47,000 and 27,000 Da polypeptides were identified as induced upon pMMO expression, with the 27,000 Da polypeptide identified as the acetylene-binding protein.
    • The enzyme complex contained 2.5 Fe and 14.5 Cu atoms per 99,000 Da.
    • EPR spectroscopy revealed type 2 copper and high-spin iron signals.
    • The purified pMMO exhibited specific activity of 11.1 nmol propylene oxidized/min/mg protein, stimulated by metal ions and inhibited by specific cytochrome b inhibitors.

    Conclusions:

    • A method for active pMMO purification was established, yielding a multi-subunit complex.
    • The purified pMMO contains specific metal cofactors essential for its catalytic activity.
    • Spectroscopic and activity data provide insights into the enzyme's mechanism and regulation.