Structural and Functional Properties of Cytochrome c Oxidase from Bacillus subtilis W23

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Abstract
The terminal component of the electron transport chain, cytochrome c oxidase (ferrocytochrome c: oxygen oxidoreductase) was purified from Bacillus subtilis W23. The enzyme was solubilized with alkyglucosides and purified to homogeneity by cytochrome c affinity chromatography. The enzyme showed absorption maxima at 414 nm and 598 nm in the oxidized form and at 443 nm and 601 nm in the reduced form. Upon reaction with carbon monoxide of the reduced purified enzyme the absorption maxima shifted to 431 nm and 598 nm. Sodium dodecylsulfate polyacrylamide gel electrophoresis indicated that the purified enzyme is composed out of three subunits with apparent molecular weights of 57 000, 37 000 and 21 000. This is the first report on a bacterial aa3-type oxidase containing three subunits. The functional properties of the enzyme are comparable with those of the other bacterial cytochrome c oxidases. The reaction catalyzed by this oxidase was strongly inhibited by cyanide, azide and monovalent salts. Furthermore a strong dependence of cytochrome c oxidase activity on negatively charged phospholipids was observed. Crossed immunoelectrophoresis experiments strongly indicated a transmembranal localization of cytochrome c oxidase.