290 W. W. Wainio 



Some of these lipids are known to participate in the cytochrome c oxidase 

 system. Wainio and Greenlees (1958) (see also Greenlees and Wainio, 1959) 

 have shown that when a soluble oxidase preparation was made by successively 

 extracting heart muscle mitochondrial fragments with deoxycholate, chelate, 

 and again deoxycholate, the activity was much reduced. Reactivation was 

 accomplished by adding the first deoxycholate extract or one of a number of 

 phosphatides (Tables 3 and 4). The following compounds were ineffective: 

 oleic acid, vitamin K^, cholesterol, DL-a-tocopherol phosphate, choline, and 

 phosphorylcholine. The activation has been verified by Hatefi (1958), who 

 found that the mitochondrial lipoprotein of Basford and Green (1959) 

 increased the oxidase activity of the green particle of Basford, Tisdale, Glenn 

 and Green (1957). The lipid-soluble form of cytochrome c described by 

 Widmer and Crane (1958) was also eff'ective. 



The role of all of these lipids remains obscure. There are, however, two 

 possibilities to be borne in mind : (1) that they are structural components of the 

 mitochondrion in the sense that they facilitate the optimal arrangement of the 

 reacting enzymes ; (2) that they are actually intermediates in electron transport. 



D. The Protein 



Very little is known about the protein of cytochrome c oxidase. Its properties 

 have not been determined because it has not been obtained in a soluble form 

 except with the aid of surface active agents, i.e. it has not been purified free 

 of lipid. 



Soluble cytochrome c oxidase has the absorption spectrum of a typical 

 protein with a maximum at 279 m/^ (Wainio, Person, Eichel and Cooperstein, 

 1951). Its molecular weight has been determined to be 75,000 by Warburg 

 (1949) from the molar extinction coefficient of the protein at 283 m/f, as 

 calculated from the photochemical dissociation of the carbon monoxide 

 compound of the reduced enzyme in yeast after correction for the absorption 

 by the carbon monoxide compound of the haem at this wavelength. Wainio, 

 Eichel and Cooperstein (1952) calculated a value of approximately 58,000 by 

 relating the uncorrected sedimentation coefficient of 5-8 x 10~^^ sec to the 

 uncorrected sedimentation coefficient for cytochrome c (1-2 X 10~^^ sec) 

 and to its molecular weight (12,000). 



REACTIONS OF CYTOCHROME C OXIDASE 

 Since cytochrome c oxidase has been shown to contain copper and to require 

 a lipid for its activity, it becomes necessary to reconsider the reactions of the 

 enzyme in the light of these new data. 



A. Reaction with Ferrocytochrome c 



Although it has been known since the early work of Keilin (1930) that ferro- 

 cytochrome c is oxidized by oxidase preparations in the presence of oxygen, 



