Fourier-transform infrared studies on azide-binding to the binuclear center of the Escherichia coli bo-type ubiquinol oxidase

Abstract

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 ( 14N 3)-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 Cu B. The band at 2062.5 cm −1 showed clear splittings upon substitution with the terminally 15N-labelled azides, indicating the Cu B 2+-N=N=N structure. Partial reduction of the oxidase with β-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 o 3+-N=N=N configuration.