Azide- and cyanide-binding to the Escherichia coli bd-type ubiquinol oxidase studied by visible absorption, EPR and FTIR spectroscopies

Cytochrome bd-type ubiquinol oxidase contains two hemes b (b(558) and b(595 )) and one heme d as the redox metal centers. To clarify the structure of t he reaction center, we analyzed Escherichia coli cytochrome bd by visible a bsorption, EPR and FTIR spectroscopies using azide and cyanide as monitorin g probes for the exogenous ligand binding site. Azide-binding caused the ap pearance of a new EPR low-spin signal characteristic of ferric iron-chlorin -azide species and a new visible absorption band at 647 nm, However, the bo und azide (N-14(3)) anti-symmetric stretching infrared band (2,010.5 cm(-1) ) showed anomalies upon N-15-substitutions, indicating interactions with su rrounding protein residues or heme b(595) in close proximity. The spectral changes upon cyanide-binding in the visible region were typical of those ob served for ferric iron-chlorin species with diol substituents in macrocycle s. However, we found no indication of a low-spin EPR signal corresponding t o the ferric iron-chlorin-cyanide complexes. Instead, derivative-shaped sig nals at g = 3.19 and g = 7.15, which could arise from the heme d(Fe3+)-CN-h eme b(595)(Fe3+) moiety, were observed, Further, after the addition of cyan ide, a part of ferric heme d showed the rhombic high-spin signal that coexi sted with the g(z) = 2.85 signal ascribed to the minor heme b(595)-CN speci es. This indicates strong steric hindrance of cyanide-binding to ferric hem e d with the bound cyanide at ferric heme b(595).