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Franziska Wegerich1, Paola Turano, Marco Allegrozzi

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Introducing positive charges in human cytochrome c (cyt c) enhances its reaction rate with superoxide radicals. This leads to more sensitive biosensors for detecting reactive oxygen species.

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Area of Science:

  • Biochemistry
  • Electrochemistry
  • Biosensor development

Background:

  • Human cytochrome c (cyt c) is an electron-transfer protein.
  • Reactive oxygen species, like superoxide radicals, play roles in cellular processes and disease.
  • Developing sensitive and selective sensors for reactive oxygen species is crucial for diagnostics.

Purpose of the Study:

  • To investigate the impact of introducing positive charges (lysines) in human cyt c on its redox properties and reaction rates with superoxide radicals.
  • To engineer enhanced cyt c mutants for improved biosensor performance.
  • To develop amperometric sensors for superoxide radical detection using modified cyt c.

Main Methods:

  • Site-directed mutagenesis was used to create 11 human cyt c mutants.
  • Proteins were characterized using UV-vis spectroscopy, circular dichroism, and NMR spectroscopy.
  • Voltammetry was employed to assess redox activity and formal redox potential.
  • Spectrophotometry was used to determine reaction rate constants with superoxide radicals.
  • Mutant-based sensors were constructed using thiol-modified gold electrodes and covalently fixed proteins.

Main Results:

  • Eleven human cyt c mutants were successfully expressed and characterized.
  • Four mutants exhibited significantly higher reaction rates with superoxide radicals compared to wild-type cyt c.
  • The E66K mutant-based electrode demonstrated superior sensitivity for superoxide detection.
  • The E66K mutant sensor maintained high selectivity and good storage stability.

Conclusions:

  • Introducing positive charges into human cyt c can enhance its reactivity with superoxide radicals.
  • Engineered cyt c mutants, particularly E66K, can serve as improved recognition elements for superoxide biosensors.
  • The developed E66K mutant-based amperometric sensor offers enhanced sensitivity, selectivity, and stability for reactive oxygen species detection.