Biochemistry (Moscow)

, Volume 74, Issue 6, pp 625–632 | Cite as

Site-directed mutagenesis of cytochrome c: Reactions with respiratory chain components and superoxide radical

  • T. Yu. Pepelina
  • R. V. Chertkova
  • T. V. Ostroverkhova
  • D. A. Dolgikh
  • M. P. Kirpichnikov
  • V. G. GrivennikovaEmail author
  • A. D. Vinogradov


Three forms of horse heart cytochrome c with specific substitutions of heme cleft surface located amino acid residues involved in specific interactions with ubiquinol:cytochrome c reductase (complex III) and cytochrome c oxidase (complex IV) were constructed, and their reactions with superoxide radical produced by NADH:ubiquinone reductase (complex I) were studied. The proteins with six (K27E/E69K/K72E/K86E/K87E/E90K and K8E/E62K/E69K/K72E/K86E/K87E) and eight (K8E/K27E/E62K/E69K/K72E/K86E/K87E/E90K) substitutions were inactive in the cytochrome c oxidase reaction, and their reduction rates by complex III were significantly lower than that seen with acetylated cytochrome c. The reduction of these modified cytochromes c under conditions where complex I generates superoxide was almost completely (about 90%) inhibited by superoxide dismutase. The genetically modified cytochromes c are useful analytical reagents for studies on superoxide generation by the mitochondrial respiratory chain. Quantitative comparison of superoxide-mediated cytochrome c reduction with hydrogen peroxide-mediated Amplex Red oxidation suggests that complex I within its native environment (submitochondrial particles) produces both superoxide (∼50%) and hydrogen peroxide (∼50%).

Key words

cytochrome c site-directed mutagenesis complex I respiratory chain superoxide radical hydrogen peroxide mitochondria 


Amplex Red


\( O_2^{\underset{\raise0.3em\hbox{$\smash{\scriptscriptstyle\cdot}$}}{ - } } \)

superoxide radical


hydroxyl radical


reactive oxygen species


submitochondrial particles


superoxide dismutase


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Copyright information

© Pleiades Publishing, Ltd. 2009

Authors and Affiliations

  • T. Yu. Pepelina
    • 1
  • R. V. Chertkova
    • 1
  • T. V. Ostroverkhova
    • 1
    • 2
  • D. A. Dolgikh
    • 1
    • 2
  • M. P. Kirpichnikov
    • 1
    • 2
  • V. G. Grivennikova
    • 3
    Email author
  • A. D. Vinogradov
    • 3
    • 4
  1. 1.Shemyakin and Ovchinnikov Institute of Bioorganic ChemistryRussian Academy of SciencesMoscowRussia
  2. 2.Department of Bioengineering, Faculty of BiologyLomonosov Moscow State UniversityMoscowRussia
  3. 3.Department of Biochemistry, Faculty of BiologyLomonosov Moscow State UniversityMoscowRussia
  4. 4.Mitoengineering CenterLomonosov Moscow State UniversityMoscowRussia

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