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 the reaction center, we analyzed Escherichia coli cytochrome bd by visible absorption, EPR and FTIR spectroscopies using azide and cyanide as monitoring probes for the exogenous ligand binding site. Azide-binding caused the appearance 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 bound azide ((14)N(3)) anti-symmetric stretching infrared band (2, 010.5 cm(-1)) showed anomalies upon (15)N-substitutions, indicating interactions with surrounding protein residues or heme b(595) in close proximity. The spectral changes upon cyanide-binding in the visible region were typical of those observed for ferric iron-chlorin species with diol substituents in macrocycles. However, we found no indication of a low-spin EPR signal corresponding to the ferric iron-chlorin-cyanide complexes. Instead, derivative-shaped signals at g = 3.19 and g = 7.15, which could arise from the heme d(Fe(3+))-CN-heme b(595)(Fe(3+)) moiety, were observed. Further, after the addition of cyanide, a part of ferric heme d showed the rhombic high-spin signal that coexisted with the g(z) = 2.85 signal ascribed to the minor heme b(595)-CN species. This indicates strong steric hindrance of cyanide-binding to ferric heme d with the bound cyanide at ferric heme b(595).
Tsubaki
M
M
Department of Life Science, Faculty of Science, Himeji Institute of Technology, Kamigoori-cho, Akou-gun, Hyogo, 678-1297, Japan. tsubaki@sci.himeji-tech.ac.jp
Mogi
T
T
Hori
H
H
D001386:Azides / Q000378:metabolism*; D001665:Binding Sites; D003486:Cyanides / Q000378:metabolism*; D003573:Cytochrome b Group; D003580:Cytochromes / Q000737:chemistry / Q000378:metabolism*; D004578:Electron Spin Resonance Spectroscopy; D045222:Electron Transport Chain Complex Proteins; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins; D010088:Oxidoreductases / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D017550:Spectroscopy, Fourier Transform Infrared
D001386:Azides; D003486:Cyanides; D003573:Cytochrome b Group; D003580:Cytochromes; D045222:Electron Transport Chain Complex Proteins; D029968:Escherichia coli Proteins; D010088:Oxidoreductases; C040077:cytochrome bd terminal oxidase complex, E coli