Purification and Characterization of the CytochromecOxidase fromRhodopseudomonas sphaeroides

Abstract

When grown aerobically in the dark, R. sphaeroides develops a respiratory chain similar to that in mitochondria and the photosynthetic apparatus is suppressed. The aa3-type cytochrome c oxidase from R. sphaeroides was purified in Triton X-100 by affinity chromatography with Sepharose 4B coupled to yeast cytochrome c. The oxidase contains 14 nmol heme a/mg protein and is composed of 3 polypeptide subunits with MW of 45,000, 37,000 and 35,000. The enzyme is highly active in the presence of detergents, with a maximal velocity of 300 s-1/mol oxidase using yeast or horse heart cytochrome c. The R. sphaeroides oxidase is cross-reactive with antibodies directed against the oxidases from Paracoccus denitrificans and Saccharomyces cerevisiae. A particularly close relationship is indicated in the case of P. denitrificans. The R. sphaeroides oxidase was incorporated into phospholipid vesicles. The resulting oxidase in these vesicles demonstrates high enzymatic activity and a respiratory control ratio of 5. Using these vesicles, no evidence for proton extrusion accompanying cytochrome c oxidation was observed. The R. sphaeroides oxidase apparently does not function as a proton pump.