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Related Experiment Videos

Site-specific cross-linking as a method for studying intramolecular electron transfer

H S Pappa1, T L Poulos

  • 1Department of Molecular Biology, University of California, Irvine 92717, USA.

Biochemistry
|May 23, 1995
PubMed
Summary

Site-directed mutagenesis created cross-linked yeast cytochrome c peroxidase and cytochrome c complexes. Complex II, mimicking a crystal structure, showed rapid intramolecular electron transfer, demonstrating functional binding orientation.

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Area of Science:

  • Biochemistry
  • Structural Biology
  • Biophysics

Background:

  • Yeast cytochrome c peroxidase and yeast cytochrome c are key proteins in electron transfer.
  • Understanding intramolecular electron transfer requires well-defined protein complexes.
  • Site-specific cross-linking enables the study of structure-function relationships.

Purpose of the Study:

  • To create site-specifically cross-linked intermolecular complexes of yeast cytochrome c peroxidase and yeast cytochrome c.
  • To investigate the relationship between the structure of these complexes and intramolecular electron transfer rates.
  • To validate a crystal structure-based model for peroxidase-cytochrome c interaction.

Main Methods:

  • Site-directed mutagenesis was employed to introduce cysteine residues into both proteins.

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  • Covalently linked intermolecular complexes were formed via engineered cysteine residues.
  • Stopped-flow spectroscopy was used to measure intramolecular electron transfer rates.

    • Main Results:

    • Two distinct complexes, Complex I and Complex II, were successfully prepared and analyzed.
    • Complex I, with a specific cysteine linkage, showed undetectable intramolecular electron transfer rates.
    • Complex II, designed to mimic a known crystal structure, exhibited rapid intramolecular electron transfer (500-800 s-1).

    Conclusions:

    • The binding orientation observed in the crystal structure of the yeast peroxidase-cytochrome c complex is competent for rapid intramolecular electron transfer.
    • Site-specific cross-linking is a viable strategy for creating homogeneous, covalently linked complexes to study electron transfer.
    • The results provide insights into the mechanism of electron transfer between cytochrome c peroxidase and cytochrome c.