Cytochrome aa3 of Rhodobacter sphaeroides as a model for mitochondrial cytochrome c oxidase

Department of Biochemistry, Michigan State University, East Lansing 48824.
Journal of Biological Chemistry (Impact Factor: 4.57). 01/1993; 267(34):24264-72.
Source: PubMed


Aerobically grown Rhodobacter sphaeroides synthesizes a respiratory chain similar to that of eukaryotes. We describe the purification of the aa3-type cytochrome c oxidase of Rb. sphaeroides as a highly active (Vmax > or = 1800 s-1), three-subunit enzyme from isolated, washed cytoplasmic membranes by hydroxylapatite chromatography and anion exchange fast protein liquid chromatography. The purified oxidase exhibits biphasic kinetics of oxidation of mammalian cytochrome c, similar to mitochondrial oxidases, and pumps protons efficiently (H+/e- = 0.7) following reconstitution into phospholipid vesicles. A membrane-bound cytochrome c is associated with the aa3-type oxidase in situ, but is removed during purification. The EPR spectra of the Rb. sphaeroides enzyme suggest the presence of a strong hydrogen bond to one or both of the histidine ligands of heme a. In other respects, optical, EPR, and resonance Raman analyses of the metal centers and their protein environments demonstrate a close correspondence between the bacterial enzyme and the structurally more complex bovine cytochrome c oxidase. The results establish this bacterial oxidase as an excellent model system for the mammalian enzyme and provide the basis for site-directed mutational analysis of its energy transducing function.

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    • "During catalytic turnover, electrons are donated first to Cu A and are then transferred consecutively to heme a and to the catalytic site consisting of heme a 3 and Cu B . The turnover activity of the R. sphaeroides CcO is ∼1600 electrons/s at pH 6.5 [10]. Spectroscopic and chemical studies have provided a reasonably clear picture of the mechanism by which O 2 is reduced to 2 H 2 O during the catalytic cycle. "
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