Infrared and EPR studies on cyanide binding to the heme-copper binuclear center of cytochrome bo-type ubiquinol oxidase from Escherichia coli. Release of a CuB-cyano complex in the partially reduced state.

Cyanide-binding to the heme-copper binuclear center of bo-type ubiquinol oxidase from Escherichia coli was investigated with Fourier transform-infrared and EPR spectroscopies. Upon treatment of the air-oxidized CN-inhibited enzyme with excess sodium dithionite, a 12C-14N stretching vibration at 2146 cm-1 characteristic of the FeO3+ C=N CuB2+ bridging structure was quickly replaced with another stretching mode at 2034.5 cm-1 derived from the FeO2+ C=N moiety. The presence of ubiquinone-8 or ubiquinone-1 caused a gradual autoreduction of the metal center(s) of the air-oxidized CN-inhibited enzyme and a concomitant appearance of a strong cyanide stretching band at 2169 cm-1. This 2169 cm-1 species could not be retained with a membrane filter (molecular weight cutoff = 10,000) and showed unusual cyanide isotope shifts and a D2O shift. These observations together with metal content analyses indicate that the 2169 cm-1 band is due to a CuB.CN complex released from the enzyme. The same species could be produced by anaerobic partial reduction of the CN-inhibited ubiquinol oxidase and, furthermore, of the CN-inhibited cytochrome c oxidase; but not at all from the fully reduced CN-inhibited enzymes. These findings suggest that there is a common intermediate structure at the binuclear center of heme-copper respiratory enzymes in the partially reduced state from which the CuB center can be easily released upon cyanide-binding.