Cyanide-reactive sites in cytochrome bd complex from E. coli

Cyanide reacts with cytochrome bd from E. coli in an 'aerobically oxidized' state (mainly, an oxygenated complex b₅₅₈³⁺b₅₉₅³⁺d²⁺-O₂), bringing about (i) decomposition of the heme d²⁺ oxycomplex (decay of the 648 nm absorption band) and (ii) extensive red shift in the Soret region accompanied by minor changes in the visible range assigned to ferric heme b₅₉₅. MCD spectra show that the Soret red shift is associated with heme b₅₅₈³⁺ high-to low-spin transition. This is the first unambiguous demonstration that heme b₅₉₅ can bind exogenous ligands. No reaction of cyanide with b₅₅₈ is observed. In about 70% of the enzyme which forms the cyano complex, the spin-state transition of b₅₉₅ decay of heme d oxycomplex match each other kinetically (k_eff ca. 0.002 s^-1 at 50 mM KCN, pH 8.1, 25°C). This points to an interaction between the two hemes. The concerted binding of cyanide to d³⁺ and b₅₉₅³⁺, perhaps as a bridging ligand, is probably rate-limited by d²⁺ oxycomplex autoxidation. In the remaining 30% of the isolated bd, there is a rapid phase of cyanide-induced b₅₉₅ spin-state transition which can be tentatively assigned to that proportion of the enzyme in which heme d is initially in the ferric rather than ferrous-oxy form.