Cytochrome aa3 of Rhodobacter sphaeroides as a model for mitochondrial cytochrome c oxidase. Purification, kinetics, proton pumping, and spectral analysis. 1992

J P Hosler, and J Fetter, and M M Tecklenburg, and M Espe, and C Lerma, and S Ferguson-Miller
Department of Biochemistry, Michigan State University, East Lansing 48824.

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.

UI MeSH Term Description Entries
D007700 Kinetics The rate dynamics in chemical or physical systems.
D008081 Liposomes Artificial, single or multilaminar vesicles (made from lecithins or other lipids) that are used for the delivery of a variety of biological molecules or molecular complexes to cells, for example, drug delivery and gene transfer. They are also used to study membranes and membrane proteins. Niosomes,Transferosomes,Ultradeformable Liposomes,Liposomes, Ultra-deformable,Liposome,Liposome, Ultra-deformable,Liposome, Ultradeformable,Liposomes, Ultra deformable,Liposomes, Ultradeformable,Niosome,Transferosome,Ultra-deformable Liposome,Ultra-deformable Liposomes,Ultradeformable Liposome
D008322 Mammals Warm-blooded vertebrate animals belonging to the class Mammalia, including all that possess hair and suckle their young. Mammalia,Mammal
D008928 Mitochondria Semiautonomous, self-reproducing organelles that occur in the cytoplasm of all cells of most, but not all, eukaryotes. Each mitochondrion is surrounded by a double limiting membrane. The inner membrane is highly invaginated, and its projections are called cristae. Mitochondria are the sites of the reactions of oxidative phosphorylation, which result in the formation of ATP. They contain distinctive RIBOSOMES, transfer RNAs (RNA, TRANSFER); AMINO ACYL T RNA SYNTHETASES; and elongation and termination factors. Mitochondria depend upon genes within the nucleus of the cells in which they reside for many essential messenger RNAs (RNA, MESSENGER). Mitochondria are believed to have arisen from aerobic bacteria that established a symbiotic relationship with primitive protoeukaryotes. (King & Stansfield, A Dictionary of Genetics, 4th ed) Mitochondrial Contraction,Mitochondrion,Contraction, Mitochondrial,Contractions, Mitochondrial,Mitochondrial Contractions
D008954 Models, Biological Theoretical representations that simulate the behavior or activity of biological processes or diseases. For disease models in living animals, DISEASE MODELS, ANIMAL is available. Biological models include the use of mathematical equations, computers, and other electronic equipment. Biological Model,Biological Models,Model, Biological,Models, Biologic,Biologic Model,Biologic Models,Model, Biologic
D008970 Molecular Weight The sum of the weight of all the atoms in a molecule. Molecular Weights,Weight, Molecular,Weights, Molecular
D010084 Oxidation-Reduction A chemical reaction in which an electron is transferred from one molecule to another. The electron-donating molecule is the reducing agent or reductant; the electron-accepting molecule is the oxidizing agent or oxidant. Reducing and oxidizing agents function as conjugate reductant-oxidant pairs or redox pairs (Lehninger, Principles of Biochemistry, 1982, p471). Redox,Oxidation Reduction
D010743 Phospholipids Lipids containing one or more phosphate groups, particularly those derived from either glycerol (phosphoglycerides see GLYCEROPHOSPHOLIPIDS) or sphingosine (SPHINGOLIPIDS). They are polar lipids that are of great importance for the structure and function of cell membranes and are the most abundant of membrane lipids, although not stored in large amounts in the system. Phosphatides,Phospholipid
D011522 Protons Stable elementary particles having the smallest known positive charge, found in the nuclei of all elements. The proton mass is less than that of a neutron. A proton is the nucleus of the light hydrogen atom, i.e., the hydrogen ion. Hydrogen Ions,Hydrogen Ion,Ion, Hydrogen,Ions, Hydrogen,Proton
D002258 Carbonyl Cyanide m-Chlorophenyl Hydrazone A proton ionophore. It is commonly used as an uncoupling agent and inhibitor of photosynthesis because of its effects on mitochondrial and chloroplast membranes. CCCP,Carbonyl Cyanide meta-Chlorophenyl Hydrazone,Carbonylcyanide 4-Chlorophenylhydrazone,Propanedinitrile, ((3-chlorophenyl)hydrazono)-,Carbonyl Cyanide m Chlorophenyl Hydrazone,4-Chlorophenylhydrazone, Carbonylcyanide,Carbonyl Cyanide meta Chlorophenyl Hydrazone,Carbonylcyanide 4 Chlorophenylhydrazone

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