Abstract
A membrane-bound cytochrome oxidase from Azobacter vinelandii was purified 20-fold using a detergent-solubilization procedure. Activity was monitored using an ascorbate-TMPD oxidation assay. The oxidase was ‘solubilized’ from a sonic-type electron-transport particle (R 3 fraction) using Triton X-100 and deoxycholate. Low detergent concentrations first solubilized the flavoprotein oxidoreductases, then higher concentrations of Triton X-100 and KCl solubilized the oxidase, which was precipitated at 27–70% (NH 4) 2SO 4. The highly purified cytochrome oxidase has a V of 60–78 μgatom O consumed/min per mg protein. TMPD oxidation by the purified enzyme was inhibited by CO, KCN, NaN 3 and NH 2OH; NaNO 2 (but not NaNO 3) also had a potent inhibitory effect. Spectral analyses revealed two major hemoproteins, the c-type cytochrome c 4 and cytochrome o; cytochromes a 1 and d were not detected. The Azotobacter cytochrome oxidase is an integrated cytochrome c 4− o complex, TMPD-dependent cytochrome oxidase activity being highest in preparations having a high c-type cytochrome content. This TMPD-dependent cytochrome oxidase serves as a major oxygen-activation site for the A. vinelandii respiratory chain. It appears functionally analogous to cytochrome a+ a 3 oxidase of mammalian mitochondria.
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