Pathways of cytochrome c oxidation by soluble and membrane-bound cytochrome aa3.
In media of low ionic strength, membraneous cytochrome c oxidase, isolated cytochrome c oxidase, and proteoliposomal cytochrome c oxidase each bind cytochrome c at two sites, one of low affinity (1 microM greater than Kd' greater than 0.2 microM) and readily reversible and the other of high affinity (0.01 microM greater than Kd) and weakly reversible. When cytochrome c occupies both sites, including the low affinity site, the maximal turnover measured polarographically with ascorbate and N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD) is independent of TMPD concentration, and lies between 250 and 400 s-1 (30 degrees C, pH 7.4) for fully activated systems. The apparent affinity of the enzyme for cytochrome c is, however, TMPD dependent. When cytochrome c occupies only the high-affinity site, the maximal turnover is closely dependent upon the concentration of TMPD, which, unlike ascorbate, can reduce bound cytochrome c. As TMPD concentration is increased, the maximal turnover approaches that seen when both sites as occupied. The lower activity of isolated cytochrome aa3 is due to the presence of inactive or inaccessible enzyme molecules. Incorporation of isolated enzyme into phospholipid vesicles restores full activity to all the subsequently accessible cytochrome aa3 molecules. Negatively charged (asolectin) vesicles show a higher cytochrome c affinity at the low-affinity sites than do the other enzyme preparations. A model for the cytochrome c-cytochrome aa3 complexes is put forward in which both sites, when occupied, are fully catalytically competent, but in which occupation of the "tight" site by a catalytically functional cytochrome c molecule is required for overall oxidation of cytochrome c via the "loose" site.
D000818:Animals; D001665:Binding Sites; D002417:Cattle; D003574:Cytochrome c Group / Q000378:metabolism*; D003576:Electron Transport Complex IV / Q000378:metabolism*; D007700:Kinetics; D008081:Liposomes; D008928:Mitochondria / Q000201:enzymology*; D008929:Mitochondria, Heart / Q000201:enzymology*; D009994:Osmolar Concentration; D010101:Oxygen Consumption; D011485:Protein Binding; D013367:Submitochondrial Particles / Q000201:enzymology*; D013772:Tetramethylphenylenediamine
D003574:Cytochrome c Group; D008081:Liposomes; D003576:Electron Transport Complex IV; D013772:Tetramethylphenylenediamine