Composition of Cytochrome c Oxidase 291 



the study of the interactions of the purified preparations has had to await the 

 isolation of a soluble cytochrome c oxidase. Wainio (1955a) has demonstrated 

 that cytochrome c oxidase (as measured at 605 m//) can be partially reduced 

 under anaerobic conditions with the simultaneous partial oxidation of ferro- 

 cytochrome c. The calculated equilibrium constants varied somewhat sug- 

 gesting that perhaps a third component was participating. This may have been 

 residual oxygen. It is also possible to oxidize reduced cytochrome c oxidase 

 partly with ferricytochrome c (Wainio, 1955b), although the equihbrium 

 attained is not the same as when oxidized oxidase and ferrocytochrome c 

 are mixed. 



With two groups to be considered, the haem and the copper, it becomes 

 necessary to assign tentatively to one of these the position of reacting with 

 ferrocytochrome c. Since the group must accept the electrons from ferro- 

 cytochrome c, and if we assume that the copper is not oxidized and reduced, 

 the role of electron acceptor falls to the haem. 



The role of the lipid remains more uncertain. It might be asked whether 

 the phosphatide activator serves to link cytochrome c and the oxidase to- 

 gether or even to transfer the electrons from one haem to the other. Further- 

 more, is its role the same or related to the role of the lipid in the lipid-soluble 

 form of cytochrome c ? 



B. Reaction with Oxygen 



By definition cytochrome oxidase is the enzyme that reduces oxygen. Again 

 it becomes necessary to ask the question whether it is the copper or the haem 

 that acts as the electron donor. As has already been pointed out, the copper 

 probably does not participate in electron transfer, by reason of its being in 

 the reduced state when the haem is oxidized, but serves merely to attach the 

 oxygen to the enzyme. The role of electron donor, as well as acceptor, would 

 then be assigned to the haem. 



Although the mechanism of the reduction is unknown, Michaelis (1951) 

 has proposed that, according to the principle of single-electron transfer, 



e 



oxygen can only be reduced in the following successive stages : Og -^ O^^ 

 -> 0^~ -^ O^^' -> Oa'*^, which in the presence of water would form the 

 following compounds with four protons : O2 > HO2 > H2O2 > HgO 



+ HO-%2H20. 



H+ ^ 



If the copper : haem ratio proves to be 2: 1, as the preliminary data show, 

 the conditions outlined above would be met. There would be two atoms of 

 cuprous copper to attach the oxygen, and one molecule of haem to act as 

 electron donor and to reduce the oxygen in successive one-electron transfers. 



Sekuzu, Takemori, Yonetani and Okunuki (1959) have postulated the 

 existence of an oxygenated form of cytochrome oxidase which is formed from 



