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.