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%).
A number of mutant forms of horse cytochrome-c with single or double substitutions of lysine residues near the heme cavity was prepared that provided an interaction of mitochondrial ubiquinone with cytochrome-c reductase (EC 1.10.2.2) (complex III) and cytochrome-c oxidase (EC 1.9.3.1) (complex IV). The succinate cytochrome-c reductase and cytochrome-c oxidase activities of mitoplasts of rat liver were measured in the presence of mutant forms of cytochrome-c. The lysine residues in positions 8, 27, 72, 86, and 87 were shown to be the main contribution to the formation of a reactive complex with ubiquinol-cytochrome-c reductase of the respiratory chain, whereas the lysine residues in positions 13, 79, 86, and 87 were predominantly responsible for the formation of a complex with cytochrome-c oxidase.
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