Cytochrome Oxidases of Pseudomonas Aeruginosa 303 



(1958a and b), and Horio et al. (1958) succeeded in highly purifying four kinds 

 of respiratory components of Pseudomonas aeruginosa. Pseudomonas (P-) 

 cytochrome-551 and P-blue protein have been crystalHzed (Horio et al, 1958; 

 Horio, 1958b), and P-cytochrome oxidase has recently been obtained in a 

 state of nearly homogeneous purity. 



The present paper deals with reactions of the animal and bacterial cyto- 

 chrome oxidases with their related respiratory components. 



Purification of Ox-heart and Pseudomonas Cytochrome Oxidases 



Ox-heart cytochrome a can be purified with the aid of cholate up to a state 

 spectrophotometrically free of the other cytochromes. Yonetani et al. 

 (1958) found that cholate inhibits the cytochrome oxidase activity of the 

 cytochrome a preparation, and that the inhibitory action can be considerably 

 diminished by the subsequent use of non-ionic detergents such as Emasol 

 4130 and Tween 80. By this method, the cytochrome oxidase activity of the 

 preparation amounts to as much as one-third of the turnover number (oxygen 

 consumed/cytochrome a) of the original extract. 



The specific activity of P-cytochrome oxidase was increased approximately 

 250 times over the first cell-free extract of Pseudomonas aeruginosa, according 

 to the method of Horio (1958a), and Horio et al. (1958), with some modi- 

 fications in which zone-electrophoresis on a vertical starch column was 

 adopted and acetone fractionation was not used. The enzyme could be 

 further purified by dialysing a concentrated sample solution against distilled 

 water, for the enzyme was remarkably less water-soluble in the absence of 

 any salt than in the presence. The purified enzyme has been found to be 

 ultracentrifugally homogeneous. 



Comparisons between Cytochrome Oxidase Activities of the Purified and 

 Non-purified Oxidase Samples 



The purified cytochrome a is easily reduced by /7-phenylenediamine, but 

 only slightly by hydroquinone, and ascorbate. Despite this fact, the cyto- 

 chrome a preparation does not consume oxygen with these reductants without 

 the addition of cytochrome c. If a suflftcient amount of cytochrome c is 

 added however, the cytochrome a preparation shows a rapid oxygen uptake 

 by the reductants, as shown in Table 1 . The same fact can be observed with a 

 particulate preparation of cytochrome oxidase which is free of cytochrome c. 

 Moreover, the cytochrome oxidase activity of the cytochrome a preparation 

 is inhibited by the typical inhibitors of cytochrome oxidase, carbon monoxide, 

 cyanide, etc., in a manner similar to that of the cytochrome oxidase activity 

 of the particulate preparation (Green and Brosteaux, 1936). Such similarities 

 are observed with respect to optimal pH of the activity and effect of oxygen 



