2Department of Bioengineering, Faculty of Biology, Lomonosov Moscow State University, 119991 Moscow, Russia; fax: (495) 939-5738; E-mail: email@example.com
3Department of Biochemistry, Faculty of Biology, Lomonosov Moscow State University, 119991 Moscow, Russia; fax: (495) 939-1376; E-mail: firstname.lastname@example.org
4Mitoengineering Center, Lomonosov Moscow State University, 119991 Moscow, Russia; fax: (495) 939-1376; E-mail: email@example.com
* To whom correspondence should be addressed.
Received November 13, 2008; Revision received December 23, 2008
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