Fourier-transform infrared studies on azide-binding to the binuclear center of the Escherichia coli bo-type ubiquinol oxidase.
Azide-binding to the heme-copper binuclear center of bo-type ubiquinol oxidase from Escherichia coli was investigated with Fourier-transform infrared spectroscopy. Deconvolution analyses of infrared spectra of the azide (14N3)-inhibited air-oxidized form showed a major infrared azide antisymmetric stretching band at 2041 cm(-1). An additional band developed at 2062.5 cm(-1) during a longer incubation. Isotope substitutions with terminally 15N-labelled azides did not show a splitting of the major band, indicating that the geometry of the bound azide is mainly in a bridging configuration between high-spin heme o and CuB. The band at 2062.5 cm(-1) showed clear splittings upon substitution with the terminally 15N-labelled azides, indicating the Cu(2+)B-N=N=N structure. Partial reduction of the oxidase with beta-NADH in the presence of azide caused an appearance of new infrared bands at 2038.5 (major) and 2009 (minor) cm(-1). The former band also showed clear splittings in the presence of the terminally 15N-labelled azides, indicating that reduction of low-spin heme b alters the structure of the binuclear center leading to the Fe(3+)o-N=N=N configuration.
Tsubaki
M
M
Department of Life Science, Faculty of Science, Himeji Institute of Technology, Hyogo, Japan. tsubaki@sci.himeji-tech.ac.jp
Mogi
T
T
Hori
H
H
D001386:Azides / Q000378:metabolism*; D003576:Electron Transport Complex IV / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D009243:NAD / Q000378:metabolism; D017550:Spectroscopy, Fourier Transform Infrared
D001386:Azides; D009243:NAD; C038862:cytochrome o oxidase; D003576:Electron Transport Complex IV