Comparison of the ligand-binding properties of native and copper-less cytochromes bo from Escherichia coli.
The binding of four anionic ligands, cyanide, fluoride, azide and formate, to cytochrome bo purified from Escherichia coli cells grown with a copper supplement (+Cu cyt.bo) is described. Membrane-bound cytochrome bo that lacks the copper component, CuB, of its active site can be prepared from cells grown under conditions where the availability of copper is limited by the presence of a CuI chelator, 2,2'-bicinchinonic acid. The ligand-binding properties of this copper-less enzyme (-Cu cyt.bo) are compared with those of +Cu cyt. bo. As judged from near-UV/visible spectroscopic changes, cyanide forms a low-spin complex with +Cu cyt.bo, whereas azide, fluoride and formate form high-spin complexes. The pH-dependences of binding suggest that for all four of these anionic ligands, both the rates of binding and the binding affinities are primarily dependent on the concentration of their protonated forms. -Cu cyt.bo, which shows less than 15% of the duroquinol oxidase activity of +Cu cyt.bo, binds cyanide, azide and fluoride, but with greatly decreased affinity (<1/30, 1/2000 and 1/2500 respectively at pH5.5 compared with +Cu cyt.bo). The complex of azide with -Cu cyt.bo still seems to be high-spin and azide binding to -Cu cyt.bo is still pH-dependent, although less so than azide binding to +Cu cyt.bo.
Moody
A J
AJ
Glynn Research Foundation, Glynn, Bodmin, Cornwall PL30 4AU, U.K.
Mitchell
R
R
Jeal
A E
AE
Rich
P R
PR
D001386:Azides / Q000378:metabolism; D003300:Copper / Q000378:metabolism*; D003486:Cyanides / Q000378:metabolism; D003573:Cytochrome b Group; D003580:Cytochromes / Q000737:chemistry / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins; D005459:Fluorides / Q000378:metabolism; D005561:Formates / Q000378:metabolism; D007700:Kinetics; D008024:Ligands; D011485:Protein Binding
D001386:Azides; D003486:Cyanides; D003573:Cytochrome b Group; D003580:Cytochromes; D029968:Escherichia coli Proteins; D005561:Formates; D008024:Ligands; D003300:Copper; C017168:cytochrome bo, E coli; D005459:Fluorides