Composition of Cytochrome c Oxidase 287 



The data showing the relationship of the copper to the haem (Wainio, 

 Vander Wende and Shimp, 1959) for a typical experiment are presented in 

 Table 1. The average ratio of copper : haem is 1-4. It is to be noted that the 

 copper: protein ratio is high in those fractions (5 and 6) which have a marked 

 absorption at 605 m/i in the reduced state (from which the haem content was 

 calculated). In another experiment (Table 2) where the copper was deter- 

 mined colorimetrically, the copper: haem ratios were found to be higher and 

 more variable (average = 2-1). However, since in these experiments the first 

 fraction was employed as an activator, rather constant ratios were obtained 

 for the activity: haem and activity : copper. 



It is not possible to conclude from these experiments whether 1 or 2 copper 

 atoms are present per haem, although the more hkely figure is 2. The averages, 

 1-4 and 2-1, are to be compared with the value of 2-3 obtained by recalculating 

 our earlier data (Eichel, Wainio, Person and Cooperstein, 1950). We find 

 that one of our partially purified preparations '2-05-2' has a ratio of 1-6 and 

 our presumed purest preparation '2-4-1-5' (Greenlees and Wainio, 1959) 

 has a ratio of 1-0. According to unpublished data of Mackler (1956) the 

 cytochrome c oxidase moiety of the electron transport particle has a copper : 

 haem ratio of 2-5, 



Vander Wende (1959) has analysed the state of the copper in cytochrome c 

 oxidase. It has been reported by Okunuki et al. (1958) that the copper is 

 firmly bound to the protein. In our experiments high concentrations 

 (0-1 m) of cyanide, diethyldithiocarbamate, ethylxanthate, etc., and prolonged 

 dialysis (up to 12 hr) were required to remove the copper and to decrease the 

 activity. The copper exists in the cuprous state in the oxidized enzyme as 

 shown by the specific reaction with biquinoline. Cupric ion was ineffective in 

 restoring the activity of preparations from which the copper had been removed 

 by dialysis against diethyldithiocarbamate. Cuprous ion was unexpectedly 

 effective in one experiment, raising the activity to 320 % of the control when 

 99 % of the copper was restored. These experiments suggest that the metal 

 does not participate in electron transport. It may function, as does the copper 

 in haemocyanin, to complex the oxygen to the enzyme. 



The spectral changes accompanying the removal of the copper were 

 inconclusive. Some reagents caused no alteration in the spectrum of the 

 reduced enzyme even though they removed much of the copper. However, 

 those that did alter the spectrum, notably cyanide, diethyldithiocarbamate and 

 ethylxanthate, caused a decrease in the absorption at 605 m/i with a shift in 

 the maximum to about 595 m,M and a decrease in absorption at 442 m/i with 

 a shift in the maximum to about 435 m/<. 



C. The Lipids 



Cytochrome c oxidase is associated with the insoluble particulate matter of 

 the cell. It is a mitochondrial enzyme which has been shown to be attached 



