Site-directed mutagenesis of cytochrome c: reactions with respiratory chain components and superoxide radical.
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%).
Pepelina
T Yu
TY
Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow, 117997, Russia.
Chertkova
R V
RV
Ostroverkhova
T V
TV
Dolgikh
D A
DA
Kirpichnikov
M P
MP
Grivennikova
V G
VG
Vinogradov
A D
AD
D000818:Animals; D045304:Cytochromes c / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D004579:Electron Transport / Q000502:physiology*; D042967:Electron Transport Complex I / Q000378:metabolism; D014450:Electron Transport Complex III / Q000378:metabolism; D003576:Electron Transport Complex IV / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism; D006736:Horses / Q000378:metabolism; D006861:Hydrogen Peroxide / Q000378:metabolism; D008099:Liver / Q000201:enzymology; D008928:Mitochondria / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000302:isolation & purification / Q000378:metabolism; D009206:Myocardium / Q000201:enzymology / Q000378:metabolism; D010084:Oxidation-Reduction; D051381:Rats; D013367:Submitochondrial Particles / Q000378:metabolism; D013482:Superoxide Dismutase / Q000378:metabolism; D013481:Superoxides / Q000378:metabolism*
D050505:Mutant Proteins; D013481:Superoxides; D045304:Cytochromes c; D006861:Hydrogen Peroxide; D013482:Superoxide Dismutase; D003576:Electron Transport Complex IV; D042967:Electron Transport Complex I; D014450:Electron Transport Complex III