1. 1. Cytochrome c oxidase (ferrocytochrome c:oxygen oxidoreductase, EC 18.104.22.168) 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 · 103 k (min-1) per mg of protein with 15 μmol 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 μmol 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. 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.
Bibliographical noteFunding Information:
The Deutscher Akademischer Austauschdienst provided R.B. with a fellowship for a short-term study at the Institute for Physiological Chemistry and Physical Biochemistry of the University of Munich where Dr. Sigurd Werner generously contributed valuable help and advice for this research. This research was supported by Public Health Service research grant GM-19398 from the National Institute of General Medical Sciences and by a Faculty Grant-in-Aid of Research from the University of Minnesota Graduate School.
- (Botryodiplodia theobromae)
- Cytochrome c oxidase structure
- Cytochrome c oxidase synthesis
- Mitochondrial biogenesis
- Spore germination