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Type 2-depleted fungal laccase.

P M Hanna1, D R McMillin, M Pasenkiewicz-Gierula

  • 1Department of Chemistry, Purdue University, West Lafayette, IN 47907.

The Biochemical Journal
|July 15, 1988
PubMed
Summary

Researchers developed new methods to remove type 2 copper from fungal laccase, revealing insights into copper centers. Restoring type 2 copper fully reactivated the enzyme, highlighting its crucial role.

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Area of Science:

  • Biochemistry
  • Bioinorganic Chemistry
  • Enzymology

Background:

  • Fungal laccase is a multi-copper oxidase crucial for lignin degradation.
  • Understanding the roles of individual copper centers (Type 1, Type 2, Type 3) is key to enzyme function.
  • Previous methods for copper removal were irreversible or failed to reconstitute the enzyme.

Purpose of the Study:

  • To develop novel methods for reversibly removing the Type 2 copper center from fungal laccase.
  • To investigate the spectral and electronic properties of copper centers upon Type 2 copper removal.
  • To elucidate the role of Type 2 copper in laccase activity and electron transfer.

Main Methods:

  • Extended dialysis against high cyanide concentrations to remove copper.
  • Development of two new reversible methods for Type 2 copper removal.
  • Spectroscopic analyses including visible absorption, circular dichroism (c.d.), and electron paramagnetic resonance (e.p.r.).

Main Results:

  • Visible absorption at 610 nm (Type 1 Cu) remained unaffected, while absorbance at 330 nm (Type 3 Cu) decreased by 60%.
  • A new absorbance increase at 435 nm was observed, assigned to a semi-reduced Type 3 copper pair.
  • E.p.r. spectra revealed well-resolved ligand hyperfine splitting for Type 1 Cu and a stable semi-reduced Type 3 site (40% of pairs).

Conclusions:

  • The Type 2 copper center can be reversibly removed, allowing for the study of other copper sites.
  • The Type 2 copper is essential for full laccase activity, with its removal leading to a stable semi-reduced Type 3 site.
  • Intramolecular electron transfer is significantly impaired in Type 2 depleted laccase, explaining the drastic loss of activity.

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