Magnetic circular dichroism used to examine the interaction of Escherichia coli cytochrome bd with ligands.
The interactions of the fully reduced and fully oxidized cytochrome bd from E. coli with ligands CO, NO, and CN- have been studied by a combination of absorption and magnetic circular dichroism (MCD) spectroscopy. In the reduced cytochrome bd, MCD resolves individual bands due to the high-spin heme b595 and the low-spin heme b558 components of the enzyme, allowing one to separately monitor their interactions along with ligand binding to the heme d component. The data show that at low concentrations, the ligands bind almost exclusively to heme d. At high concentrations, the ligands begin to interact with the low-spin heme b558. At the same time, no evidence for significant binding of the ligands to the high-spin heme b595 is revealed in either the reduced or the fully oxidized cytochrome bd complex. The data support the model [Borisov, V. B., Gennis, R. B., and Konstantinov, A. A. (1995) Biochemistry (Moscow) 60, 231-239] according to which the two high-spin hemes d and b595 share a high-affinity ligand binding site with a capacity for only a single molecule of the ligand; i.e., there is a strong negative cooperativity with respect to ligand binding to these two hemes with cytochrome d having an intrinsic ligand affinity much higher than that of heme b595.
Borisov
V
V
A. N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, Russia.
Arutyunyan
A M
AM
Osborne
J P
JP
Gennis
R B
RB
Konstantinov
A A
AA
D001665:Binding Sites; D002248:Carbon Monoxide / Q000737:chemistry; D002942:Circular Dichroism; D003485:Cyanates / Q000737:chemistry; D003573:Cytochrome b Group; D003580:Cytochromes / Q000737:chemistry*; D045222:Electron Transport Chain Complex Proteins; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins; D006418:Heme / Q000737:chemistry; D008024:Ligands; D008280:Magnetics; D008956:Models, Chemical; D009569:Nitric Oxide / Q000737:chemistry; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000737:chemistry*; D013696:Temperature
D003485:Cyanates; D003573:Cytochrome b Group; D003580:Cytochromes; D045222:Electron Transport Chain Complex Proteins; D029968:Escherichia coli Proteins; D008024:Ligands; D009569:Nitric Oxide; D006418:Heme; D002248:Carbon Monoxide; D010088:Oxidoreductases; C040077:cytochrome bd terminal oxidase complex, E coli