Cytochrome aa3 of Rhodobacter sphaeroides as a model for mitochondrial cytochrome c oxidase. Purification, kinetics, proton pumping, and spectral analysis.
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.
Hosler
J P
JP
Department of Biochemistry, Michigan State University, East Lansing 48824.
Fetter
J
J
Tecklenburg
M M
MM
Espe
M
M
Lerma
C
C
Ferguson-Miller
S
S
D000818:Animals; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone / Q000494:pharmacology; D002462:Cell Membrane / Q000201:enzymology; D002845:Chromatography; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D017886:Durapatite; D004578:Electron Spin Resonance Spectroscopy; D003576:Electron Transport Complex IV / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D006882:Hydroxyapatites; D007700:Kinetics; D008081:Liposomes; D008322:Mammals; D008928:Mitochondria / Q000201:enzymology*; D008954:Models, Biological; D008970:Molecular Weight; D010084:Oxidation-Reduction; D010743:Phospholipids; D011522:Protons; D012242:Rhodobacter sphaeroides / Q000201:enzymology*; D013053:Spectrophotometry; D013059:Spectrum Analysis, Raman
D006882:Hydroxyapatites; D008081:Liposomes; D010743:Phospholipids; D011522:Protons; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone; D017886:Durapatite; D003576:Electron Transport Complex IV