Cytochrome bd oxidase from XXXX . Purification and quantitation of ligand binding to the @entity26 reduction site.

@entity73184 @entity26 @entity11072 @entity3789 @entity920 @entity1486 @entity68 @entity1446
Cytochrome bd has been purified from @entity1486 by a new simplified procedure. The @entity1446 and total @entity68 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 @entity1446 d only, with a stoichiometry of 1 molecule of gas per 2 molecules of @entity1446 b or per 3 atoms of @entity68 . The results clearly define a stoichiometry of one @entity1446 d per complex. Low affinity binding of CO and NO to @entity1446 b595 also occurs at higher ligand concentrations. EPR @entity1446 - @entity11072 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 @entity26 from @entity1446 d and a change in line shape of the high spin region of the EPR spectrum. @entity3789 binds to both @entity1446 d and @entity1446 b595 in the air-oxidized complex, displacing molecular @entity26 from @entity1446 d. The rate of @entity3789 binding to @entity1446 d as assessed by spectral changes at 650 nm does not correlate with the rate of binding to @entity73184 as assessed by the loss of the high spin EPR signal. In addition, the @entity3789 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 @entity920 -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.
