J. Biochem, 1992, Vol. 112, No. 6 816-821
© 1992 Japanese Biochemical Society
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Thiobacillus ferrooxidans Cytochrome c Oxidase: Purification, and Molecular and Enzymatic Features
Department of Life Science, Faculty of Bioscience and Biotechnology, Tokyo Institute of Technology Nagatsuta, Midori-ku, Yokohama 227
2To whom correspondence should be addressed
Cytochrome c oxidase from Thiobacillus ferrooxidans was purified to homogeneity and some of its properties were studied. The oxidase was solubilized with n-octyl-ß-D-thioglucoside (OTG) under acidic conditions (pH 4.0) and purified by one step of ionexchange chromatography with a CM-Toyopearl column. The absorption spectrum of the oxidase showed peaks at 420 and 898 nm in the oxidized form and at 440 and 595 nm in the reduced form. Its CO compound showed a novel absorption spectrum; a double-peaked 
band appeared at 429 and 438 nm. The oxidase seemed to have CuA -like copper atom from its ESR and near-infrared spectra. The oxidase molecule consisted of three polypeptides with molecular weights of 53,000, 22,000, and 17,000, respectively, as estimated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The molecular weight of the enzyme in a solution containing detergents was estimated to be 169,000 on the basis of the results obtained by gel filtration, while the molecular weight per heme 
was estimated to be 83,700. The copper content of the oxidase was 1.01 g atom per mol of heme . Therefore, the cytochrome seemed to contain one molecule of heme and one atom of copper in the minimal structural unit consisting of one molecule each of the three subunits, and to occur as a dimer of the unit in the solution. The oxidase oxidized ferrocytochrome c-552 of the bacterium, and the optimal pH of the reaction was 3.5. The enzyme also oxidized the reduced form of rusticyanin, and the optimum pH of the reaction was 4.0. In the enzymatic oxidation of ferrocytochrome c-552 at pH 4.0, Km of the oxidase for the ferrocytochrome was determined to be 17 µM, and Vmax of the enzyme to be 6.3 mol of ferrocytochrome c oxidized per mol of heme a of the oxidase per s. In the oxidation of the reduced form of rusticyanin at pH 4.0, Km of the oxidase for the protein was approximately 600 pM and Vmax of the enzyme was approximately 50 mol of reduced rusticyanin oxidized per mol of heme of the oxidase per s. Sulfate ions stimulated the enzymatic oxidation of these electron donors. It seems interesting that -type cytochrome c oxidase can oxidize a copper protein at a high molecular activity.
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