366 VIII. HEMATIN ENZYMES, I. CYTOCHROME SYSTEM 



the catalase of the bacterial cell, allowing hydrogen peroxide to oxidize the 

 protohemochrome of cytochrome b to a hemzchrome with an oxyporphyrin 

 ring. The pyridine hemichrome of oxyporphyrin has a band at 639 mfx. 



Keilin and Harpley {lUSJf) found no oxidation of cytochrome c by crushed 

 Escherichia coli, although E. coli contains a trace of cytochrome ai. While 

 the identity of cytochrome a i with the respiratory ferment of A. pasteurianum 

 is probable, it cannot be considered definitely established. 



3.6.4. Cytochrome Oxidase. In washed heart muscle and other 

 tissues Keilin {11^76) discovered an enzyme which catalyzed the 

 oxidation of p-phenylenediamine, or a mixture of dimethyl-p-phenyl- 

 enediamine with a-naphthol, the so-called Nadi reagent {653,2327). 

 He called it indophenol oxidase. Later Keilin and Hartree {1^77, 

 14.91) and Stotz and co-workers {2681) recognized that cytochrome c 

 was required for the oxidation of p-phenylenediamine, hydroqui- 

 none, and cysteine, ferricytochrome c oxidizing these substrates 

 and ferrocytochrome c being in turn oxidized by the oxidase. The 

 enzyme was therefore renamed cytochrome oxidase. In fact, p-phenyl- 

 enediamine is not a very suitable substrate for the cytochrome 

 oxidase-cytochrome c system, since it is partly oxidized by cyto- 

 chrome b without the oxidase {2681). 



Cytochrome oxidase is a heat-labile enzyme destroyed by heating 

 to 52° C. It is inhibited by cyanide, sulfide, azide, hydroxylamine, 

 and by carbon monoxide. The distribution coefficient, K, of Keilin's 

 cytochrome oxidase from sheep's heart (studied with cysteine as 

 substrate) is not significantly different from that of Warburg's res- 

 piratory ferment in yeast or Melnick's oxidase from rat heart. Keilin 

 found 5-10, Warburg, 9, and Melnick, 6.3. 



For a time Keilin assumed that cytochrome oxidase was a copper-con- 

 taining enzyme similar to polyphenol oxidase {1^92). This view he later 

 abandoned. Although decisive evidence is missing, it is likely that the 

 photochemical absorption spectrum measured by Melnick {1907,1909) is 

 that of cytochrome oxidase. Studies on the photochemical absorption 

 spectrum of the reconstituted cytochrome oxidase -cytochrome system 

 acting on substrates like hydroquinone or cysteine, and a comparison with 

 the photochemical spectrum of Melnick would be of interest. In such experi- 

 ments the oxidase should be prepared from rat heart, since this was used by 

 Melnick. Too little attention has been paid hitherto to the possibility of 

 species differences between the respiratory ferments 



While the enzyme can be isolated from the cells, it is still doubtful whether 

 homogeneous solutions have been obtained. The earlier preparations {1^77, 

 1491) consisted of suspensions of washed cells. Later, turbid extracts with 

 sodium phosphate were prepared. These can be liberated from accompanying 



