Mitochondrial biogenesis during fungal spore germination. Purification, properties and biosynthesis of cytochrome c oxidase from Botryodiplodia theobromae.
1. Cytochrome c oxidase (ferrocytochrome c:oxygen oxidoreductase, EC 1.9.3.1) was purified from the mycelial fungus Botryodiplodia theobromae by sodium cholate and ammonium sulfate solubilization, ammonium sulfate fractionation, and DEAE-cellulose chromatography. The purified enzyme contained 6--7 nmol heme a/mg of protein. The specific activity of the purified enzyme was 2.1--2.3 . 10(3) k (min-1) per mg of protein with 15 mumol ferrocytochrome c and at pH 5.9 and optimal phosphate and Tween 80 concentrations (65 mM and 0.1%, respectively). The Km for ferrocytochrome c was determined to be 1.2--1.3 . 10(-5) M, while at infinite substrate concentration the enzyme catalyzed the oxidation of about 60 mumole of ferrocytochrome c/min per mg of protein. Sedimentation behavior and kinetic evidence suggest that the purified enzyme exists as aggregates of the single molecule. The purified B. theobromae cytochrome c oxidase with its 428 nm Soret absorption maximum may be similar if not identical to oxygenated forms of the enzyme from other fungal species. 2. The purified enzyme was shown by sodium dodecyl sulfate polyacrylamide gel electrophoresis to consist of seven polypeptides with the following respective molecular weights: I, 41 000; II, 28 000; III, 19 000; IV, 14 800; V, 12 800; VI, 11 500; and VII, 9300. Biosynthesis studies showed that the three highest molecular weight polypeptides of the enzyme were synthesized on mitochondrial ribosomes, and the four smaller polypeptides were products of cytoplasmic ribosomes.
D003574:Cytochrome c Group / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D003576:Electron Transport Complex IV / Q000032:analysis / Q000378:metabolism*; D007700:Kinetics; D008928:Mitochondria / Q000378:metabolism; D003904:Mitosporic Fungi / Q000201:enzymology*; D008970:Molecular Weight; D011136:Polysorbates; D012270:Ribosomes / Q000378:metabolism; D013057:Spectrum Analysis; D013172:Spores, Fungal / Q000378:metabolism
D003574:Cytochrome c Group; D011136:Polysorbates; D003576:Electron Transport Complex IV