INFRARED AND EPR STUDIES ON CYANIDE BINDING TO THE HEME-COPPER BINUCLEAR CENTER OF CYTOCHROME BO-TYPE UBIQUINOL OXIDASE FROM ESCHERICHIA-COLI

Cyanide-binding to the heme-copper binuclear center of bo-type ubiquin ol oxidase from Escherichia cell was investigated with Fourier transfo rm-infrared and EPR spectroscopies. Upon treatment of the air-oxidized CN-inhibited enzyme with excess sodium dithionite, a C-12-N-14 stretc hing vibration at 2146 cm(-1) characteristic of the Fe-O(3+)-C=N-Cu-B( 2+) bridging structure was quickly replaced with another stretching mo de at 2034.5 cm(-1) derived from the Fe-O(2+)-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 concomi tant appearance of a strong cyanide stretching band at 2169 cm(-1). Th is 2169 cm(-1) species could not be retained with a membrane filter (m olecular weight cutoff = 10,000) and showed unusual cyanide isotope sh ifts and a D2O shift. These observations together with metal content a nalyses indicate that the 2169 cm(-1) band is due to a Cu-B . CN compl ex released from the enzyme. The same species could be produced by ana erobic partial reduction of the CN-inhibited ubiquinol oxidase and, fu rthermore, of the CN-inhibited cytochrome c oxidase; but not at all fr om the fully reduced CN-inhibited enzymes. These findings suggest that there is a common intermediate structure at the binuclear center of h eme-copper respiratory enzymes in the partially reduced state from whi ch the Cu-B center can be easily released upon cyanide-binding.