Photoaffinity labeling of yeast cytochrome oxidase with arylazido cytochrome c derivatives

Abstract

The interaction of cytochrome c with cytochrome c oxidase, and the electron transfer occurring in the complex, are important events in the mitochondrial respiratory chain, leading to oxygen reduction, asymmetric proton release and conservation of the free energy of the redox reaction as an electrochemical gradient of hydrogen ions [l-3] . Among the approaches utilized to elucidate the structure of the cytochrome oxidase-cytochrome c complex, crosslinking of the components of the system has been recently employed by a number of investigators. Using yeast cytochrome c, modified at its free sulihydryl group by the reaction with 5,5’-dithiobis (2-nitrobenzoate), subunit III of yeast cytochrome c oxidase was indicated [4] to be close to the binding site for cytochrome c. Using horse heart cytochrome c labeled with a 3nitrophenylazido group at lysine 13, evidence was provided [5,6] that subunit II was the subunit nearest to cytochrome c in the complex with the beef-heart oxidase. This conclusion was also supported by [7] in which dithiobissuccininidylpropionate was used as crosslinking reagent. Using an azidocytochrome c derivative, the binding site of cytochrome c was found [S] to occur in the two smallest