Cytochrome o, solubilized from the membrane of Azotobacter vinelandii, has been purified to homogeneity as judged by ultracentrifugation and polyacrylamide gel electrophoresis. The detergent-containing cytochrome o is composed of one polypeptide chain with a molecular weight of 28 000-29 000, associated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The enzyme exists as a dimer by gel filtration analysis. The amino analysis which reveals the majority of residues are of hydrophobic nature. The cytochrome o oxidase contains protoheme as its prosthetic group and about 20-40% of phospholipids. The phospholipids are identified as phosphatidylethanolamine and phosphatidylglycerol by radioautographic analysis using 2-dimensional thin-layer chromatography. No copper or nonheme iron can be detected in the purified oxidase preparation by atomic absorption and chemical analyses. Oxidation-reduction titration shows this membrane-bound cytochrome o to be a low-potential component, and Em was determined to be -18 mV in the purified form and -30 mV in the membrane-bound form. Both forms bind CO with a reduced absorption peak at 559 and 557-558 nm in the native and solubilized forms, respectively. A high-spin (g = 6.0) form is assigned to the oxidized cytochrome o by electron paramagnetic resonance analysis, and KCN abolishes this high-spin signal. CO titration of purified cytochrome o in the anaerobic conditions shows the enzyme binds one CO per four protohemes and a dissociation constant is estimated to be 3.2 microM for CO. Cyanide reacts with purified cytochrome o in both oxidized and CO-bound forms, identified by specific spectral compounds absorbed at the Soret region. Cytochrome c, often co-purified with cytochrome c from the membrane, cannot serve as a reductant for cytochrome o in vitro, due to the apparent potential difference of about 300 mV. Upon separation, both cytochrome o and cytochrome c4 show a great tendency of aggregation. Furthermore, the oxidase activity (measured by tetramethyl-p-phenylenediamine oxidation rate) decreases as the cytochrome c concentration is decreased by ammonium sulfate fractionation. All these suggest the structural and functional complex nature of cytochrome c4 and cytochrome o in the membrane of A. vinelandii.