316 



Discussion 



The Reaction of Cytochrome c Oxidase with Oxygen 



The Oxygen-reducing Equivalents of Cytochromes a and a^ 



By B. Chance (Philadelphia) 



Chance: It is of considerable interest to know how many oxygen-reducing equivalents 

 are contained in cytochrome oxidase (a + 03), and L. Smith and I have contemplated 

 this problem for some time. More recently Yonetani and I (Chance and Yonetani, 

 Fed. Proc. 18, 202, 1959) have obtained spectrophotometric titrations of reduced 



0.050n 



I I I I I r I I I I I I I 



10 20 30 



>iM Fe a_ 



' I ■ I ' I ' I ' I 

 10 20 30 40 50 



>iMFe a. 



® 



Fig. 1 , An example of a spectrophotometric titration of cytochromes a, a^ 

 and c with 7-8 i-iu oxidizing equivalents. 



cytochrome oxidase by oxygen, and Yonetani and Nakamura have carried out 

 independent magnetometric titrations. 



The procedure we have used simulates enzymic activity of the oxidase. Substrate 

 is added to a concentrated solution of the oxidase and after the dissolved oxygen has 

 been exhausted and the oxidase is reduced, a small volume of air-saturated buffer is 

 rapidly mixed by means of a modified Hartridge-Roughton flow apparatus (regener- 

 ative flow apparatus) and the maximal extent of oxidation of the oxidase is recorded 

 in a few tenths of a second by spectrophotometric (double-beam spectrophotometer) 

 or magnetometric (modified Rankine balance) methods. 



In experiments with the rapid flow apparatus, it has been observed that upon 

 addition of oxygen the absorption bands at 605 m/< and 444 m/< disappear even 

 though cytochrome c is absent. Apparently cytochrome c is needed for the reduction 

 of the oxidase, not for its oxidation as has been inferred above; the rapid oxidation 

 of cytochromes a and ^3 is observable with our technique without the addition of 

 cytochrome c. 



The added oxygen is slowly expended in enzymic activity and the experiment is 

 repeated with a more dilute solution of the oxidase. The experiment is begun with 

 an excess of oxidase over oxygen and concentrations of the former are diminished 

 until there is considerably more oxygen than oxidase. The utilization of oxygen due 



