Cytochrome bd oxidase from Azotobacter vinelandii. Purification and quantitation of ligand binding to the oxygen reduction site.
Cytochrome bd has been purified from Azotobacter vinelandii by a new simplified procedure. The heme and total iron content has been measured, as has the number of high affinity CO and NO binding sites. Spectral changes indicate high affinity binding of CO and NO to heme d only, with a stoichiometry of 1 molecule of gas per 2 molecules of heme b or per 3 atoms of iron. The results clearly define a stoichiometry of one heme d per complex. Low affinity binding of CO and NO to heme b595 also occurs at higher ligand concentrations. EPR heme-nitrosyl signals are seen with NO bound to both hemes b595 and d but with no indication of spin exchange coupling. Exposure of the air-oxidized complex to alkaline pH results in removal of molecular oxygen from heme d and a change in line shape of the high spin region of the EPR spectrum. Cyanide binds to both heme d and heme b595 in the air-oxidized complex, displacing molecular oxygen from heme d. The rate of cyanide binding to heme d as assessed by spectral changes at 650 nm does not correlate with the rate of binding to heme b595 as assessed by the loss of the high spin EPR signal. In addition, the cyanide binding rate in the presence of reductant is only 3 times that of the rate of binding to the air-oxidized enzyme, in contrast to the copper-containing oxidases where strong redox cooperativity makes these two rates differ by a factor of at least 10(6). The results do not support the idea of the presence of two strongly interacting hemes in a binuclear center.
Jünemann
S
S
Metals in Biology and Medicine Centre, King's College London, United Kingdom.
Wrigglesworth
J M
JM
D016948:Azotobacter vinelandii / Q000201:enzymology*; D001665:Binding Sites; D002248:Carbon Monoxide / Q000378:metabolism; D003486:Cyanides / Q000378:metabolism; D003573:Cytochrome b Group; D003580:Cytochromes / Q000302:isolation & purification / Q000378:metabolism*; D004578:Electron Spin Resonance Spectroscopy; D045222:Electron Transport Chain Complex Proteins; D029968:Escherichia coli Proteins; D006418:Heme / Q000031:analogs & derivatives / Q000032:analysis; D007700:Kinetics; D008024:Ligands; D009569:Nitric Oxide / Q000378:metabolism; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000302:isolation & purification / Q000378:metabolism*; D010100:Oxygen / Q000378:metabolism; D013053:Spectrophotometry
D003486:Cyanides; D003573:Cytochrome b Group; D003580:Cytochromes; D045222:Electron Transport Chain Complex Proteins; D029968:Escherichia coli Proteins; D008024:Ligands; D009569:Nitric Oxide; D006418:Heme; C080647:heme d; D002248:Carbon Monoxide; D010088:Oxidoreductases; C040077:cytochrome bd terminal oxidase complex, E coli; D010100:Oxygen