318 Discussion 



The usefulness of these data depends upon the accuracy with which the extinction 

 coefficients for cytochromes a and a^ are known. The latter is known fairly accurately 

 from photodissociation kinetics (Chance, J. biol. Chem. 202, 407, 1953). The value 

 for cytochrome a has been based on an analogy with other haemoproteins, and this 

 value has more recently been found to check fairly well with chemical determinations 

 in Okunuki's laboratory. The accuracy of these determinations is sufficient to indicate 

 that electron donation from copper is rendered extremely unlikely. The data further 

 raise the question of whether cytochrome a itself has an electron donating capacity : 

 the optical constants are almost, but not quite, secure enough to justify a conclusion 

 on this point. 



Wainio: With reference to Chance's observations, the calculations are based on the use 

 of the a-peak for one compound and the y-peak for the other. If one were to average 

 the two values, in this instance would not the oxidizing equivalents added and found 

 be identical ? This would indicate that only one, not two, enzymes are involved. 



Chance: The idea that we are over-estimating the oxidizing equivalents by taking the a and 

 y bands of the same haemoprotein as separate entities is another way of stating that 

 cytochromes a and 03 are identical, and this has already been discussed by Slater and 

 by me. 



Lemberg: As a first approximation Chance assumed that the absorption change of the 

 a-band at 605 m/< is entirely due to the oxidation of cytochrome a, that of the Soret 

 band at 445 m/t to the oxidation of cytochrome a^. Our studies of the model haemo- 

 proteins a make this appear unlikely, and Chance and Yonetani {Fed. Proc. 18, 202, 

 1959) have also found that the ratios y/a for the two cytochromes are not so widely 

 different as had previously been assumed. 



If we make the more likely assumption that cytochromes a and a^ contribute almost 

 equally to the a-band, while the contribution of Og to the y-band is twice that of 

 cytochrome a, and if we assume that the concentrations of both cytochromes are 

 approximately equal, we obtain : 



'a' = a -1- ^3, '^3' = a^+\a (1) 



and 'a + a^' = l-75(a + a^ (2) 



where 'a', '^3' and 'a + 03' mean the values calculated by Chance, and a, 03 and 

 a + a^ the true values. The ratio 'a + a^'la + a^ also depends on the choice of the 

 extinction coefficients and on the aja^ ratio, but the values assumed for them are 

 probably not very far from the correct ones. 



Our earlier estimation of the porphyrin a content of ox heart muscle agreed well 

 with the values of Chance for 'a + Qz, but we may also have overestimated the 

 porphyrin a content by assuming at that time a value of 20 for the specific extinction 

 of band III of neutral porphyrin a instead of the correct value of 27. 

 Chance (note added in proof): It is clear that the interpretation of our results on the 

 titration of cytochrome oxidase with oxygen depends upon the numerical values of the 

 optical constants, and an interpretation of their meaning. In the experiment described 

 above, the concentration of cytochrome oxidase was determined by dividing 

 A£605_63o(red-ox) by 16cm~^ x mM~^; the concentration thus obtained was inter- 

 preted to be equal to that of cytochrome a alone. Thus a preparation containing 

 equal amounts of cytochromes a and 03 would accept twice as many oxidizing 

 equivalents. It follows from this that the value of Afgos-eso for calculating total 

 haematin is 8 cm"^ x mM~^. This may be converted to Ke^Q-JjQd-ox) of 7-3. This 

 value may now be compared with those obtained from determinations of the total 

 haematin content of cytochrome oxidase preparations of considerable degrees of 

 purification. For example, the value currently used in this laboratory (T. Yonetani, 

 personal communication) is 1 1 cm~^ x mM~^, and the value currently used at the 

 Enzyme Institute (D. Griffith, personal communication) is 90-9-5 cm~^ x mM~^. 

 It is important to note that none of these determinations of concentration depends 

 upon the proportion of cytochromes a and 03 which may be evaluated at 605 m/< ; 

 the total oxidase content is evaluated. 



