Purification and characterization of cytochrome O from Azotobacter vinelandii

The membrane-bound cytochrome o has been solubilized from the Azotobacter vinelandii electron transport particle and further purified by use of conventional chromatographic procedures. The cytochrome purified as a detergent (Triton X-100) and hemoprotein complex, contained 1.2 nmoles heme per mg of protein. Cold-temperature spectrum showed that no other cytochrome was associated with the purified preparation, and electrophoresis revealed that only one type of hemoprotein was obtained. The purified cytochrome o reacted with both carbon monoxide and cyanide readily. Only in the reduced form did it combine with carbon monoxide, whereas the oxidized form reacted with cyanide. An "oxygenated" form of the cytochrome £ was demonstrated to be spectrally distinguishable from both the oxidized and the reduced forms. The prosthetic group for cytochrome (D was found to be protoheme IV and the minimum molecular weight, based on protoheme content, was calculated to be 625,000 assuming one mole of heme per mole of protein. The extinction coefficients for cytochrome o were calculated on the basis of the protohemochromogen concentration and the values obtained are reported. The extinction coefficient for the CO:reduced minus reduced -1 -1 cytochrome £, A416 nm (peak) - 431 nm (trough) was 235.4 mM cm Amino acid analyses revealed the purified cytochrome o_ to be (a) an acid protein, (b) similar to cytochrome p-450 in amino acid composition, and (c) contained high concentrations of hydrophobic residues, indicating a possible structural relationship with membrane phospholipids. Lipid analyses on the cytochrome o revealed the presence of a high concentration of phospholipid; the amount of phospholipid present was 40.5% by weight. Radioautographic analyses, using two dimensional thin layer chromatography, reveal the presence of phosphatidylethanolamine and phosphatidylglycerol.