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Related Experiment Videos

Conversion of desulforedoxin into a rubredoxin center

L Yu1, M Kennedy, C Czaja

  • 1Section of Hematology Research, Mayo Clinic and Foundation, Rochester, Minnesota 55905, USA.

Biochemical and Biophysical Research Communications
|February 24, 1997
PubMed
Summary

Mutating desulforedoxin by inserting residues between cysteines altered its iron-sulfur cluster. The resulting spectroscopic properties became similar to rubredoxin, suggesting structural changes influence the iron center.

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Area of Science:

  • Biochemistry
  • Bioinorganic Chemistry
  • Protein Engineering

Background:

  • Rubredoxin and desulforedoxin are iron-sulfur proteins containing an Fe(S-Cys)4 center.
  • Spectroscopic properties of desulforedoxin's iron center differ from rubredoxin.
  • This difference is hypothesized to stem from metal site distortion due to adjacent cysteine residues in desulforedoxin.

Purpose of the Study:

  • To investigate the role of adjacent cysteine residues in desulforedoxin's unique spectroscopic properties.
  • To engineer desulforedoxin mutants to alter the metal site environment.
  • To compare the spectroscopic characteristics of engineered desulforedoxin with wild-type and rubredoxin.

Main Methods:

  • Site-directed mutagenesis to insert residues (Glycine or Pro-Val) between adjacent cysteines in desulforedoxin.

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  • UV-Vis absorption spectroscopy to analyze optical properties.
  • X-band Electron Paramagnetic Resonance (EPR) spectroscopy at low temperatures to characterize the iron center.

    • Main Results:

    • Mutant desulforedoxins exhibited distinct optical spectra with maxima at 278, 345, 380, 480, and 560 nm.
    • EPR spectra revealed high-spin Fe3+ ions with significant rhombic distortions (E/D = 0.21-0.23) in the mutants.
    • These spectroscopic features of the mutants were markedly different from wild-type desulforedoxin and closely resembled those of rubredoxin.

    Conclusions:

    • Insertion of residues between adjacent cysteines in desulforedoxin disrupts the hypothesized distortion mechanism.
    • The engineered desulforedoxin mutants adopt spectroscopic properties similar to rubredoxin.
    • This suggests that the local cysteine arrangement is critical for the distinct electronic structure of the desulforedoxin iron center.