Isolation and purification of the cytochrome oxidase of Azotobacter vinelandii.
A membrane-bound cytochrome oxidase for Azobacter vinelandii was purified 20-fold using a detergent-solubilization procedure. Activity was monitored using as ascorbate-TMPD oxidation assay. The oxidase was 'solubilized' from a sonic-type electron-transport particle (R3 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% (NH4)2SO4. The highly purified cytochrome oxidase has a V of 60-78 microgatom O consumed/min per mg protein. TMPD oxidation by the purified enzyme was inhibited by CO, KCN, NaN3 and NH2OH; NaNO2 (but not NaNO3) also had a potent inhibitory effect. Spectral analyses revealed two major hemoproteins, the c-type cytochrome c4 and cytochrome o; cytochromes a1 and d were not detected. The Azotobacter cytochrome oxidase is an integrated cytochrome c4-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+a3 oxidase of mammalian mitochondria.
D001395:Azotobacter / Q000201:enzymology*; D003573:Cytochrome b Group; D003580:Cytochromes / Q000032:analysis; D003840:Deoxycholic Acid; D003576:Electron Transport Complex IV / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D029968:Escherichia coli Proteins; D017830:Octoxynol; D011092:Polyethylene Glycols; D012995:Solubility; D013057:Spectrum Analysis; D013772:Tetramethylphenylenediamine / Q000378:metabolism
D003573:Cytochrome b Group; D003580:Cytochromes; D029968:Escherichia coli Proteins; D003840:Deoxycholic Acid; D011092:Polyethylene Glycols; D017830:Octoxynol; C017168:cytochrome bo, E coli; D003576:Electron Transport Complex IV; D013772:Tetramethylphenylenediamine