Site-Directed Mutagenesis of Cytochrome c: Reactions with Respiratory Chain Components and Superoxide Radical

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T. Yu. Pepelina¹, R. V. Chertkova¹, T. V. Ostroverkhova^1,2, D. A. Dolgikh^1,2, M. P. Kirpichnikov^1,2, V. G. Grivennikova^3*, and A. D. Vinogradov^3,4

¹Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, ul. Miklukho-Maklaya 16/10, 117997 Moscow, Russia; fax: (495) 335-2888; E-mail: rita@nmr.ru

²Department of Bioengineering, Faculty of Biology, Lomonosov Moscow State University, 119991 Moscow, Russia; fax: (495) 939-5738; E-mail: dolgikh@nmr.ru

³Department of Biochemistry, Faculty of Biology, Lomonosov Moscow State University, 119991 Moscow, Russia; fax: (495) 939-1376; E-mail: vgrivennikova@mail.ru

⁴Mitoengineering Center, Lomonosov Moscow State University, 119991 Moscow, Russia; fax: (495) 939-1376; E-mail: adv@biochem.bio.msu.su

^* To whom correspondence should be addressed.

Received November 13, 2008; Revision received December 23, 2008

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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%).

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KEY WORDS: cytochrome c, site-directed mutagenesis, complex I, respiratory chain, superoxide radical, hydrogen peroxide, mitochondria

DOI: 10.1134/S0006297909060066

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