Composition of Cytochrome c Oxidase 



297 



reduced enzyme, it is suggested that cyanide combines principally with the 

 haem and oxygen with the copper. 



Not only has Vander Wende (1959) demonstrated that cyanide will remove 

 the copper, but it is known that other copper-containing four-electron 

 transfer oxidases are sensitive to cyanide. The fact that the cyanide will only 



JO 

 Mimifes 



Fig. 5. Rate of oxidation of reduced cytochrome c oxidase by air in the presence 



of cyanide. 



remove the copper with difficulty in our experiments, suggests that the copper 

 is not too readily available to the cyanide. 



Whether the copper is a component to be considered in relationship to the 

 phenomenon presented in Fig. 6 (Wainio, 1956), can only be guessed at. 

 If cytochrome c oxidase and a small amount of ferrocytochrome c (to reduce 

 the oxidase) are incubated for 10 min with a range of concentrations of 

 cyanide before the activity is determined spectrophotometrically by adding a 

 substrate amount of ferrocytochrome c, the inhibitions observed in Fig. 6 

 are recorded. Preincubation of the oxidase alone with the cyanide, or of 

 ferricytochrome c (which is the form of cytochrome c which combines with 

 cyanide) alone with the cyanide, does not give these experimental points which 

 lie on a doubly-inflected curve. The curve is indicative of two binding-sites 

 for cyanide. One complex has a dissociation constant of approximately 

 3 X 10"^ moles/1, and the other a constant of approximately 5 x 10~^ moles/1. 

 The curve (dashes) is a theoretical curve drawn on the assumption that each 



