312 Discussion 



HoRio: The absoq)tion spectra in the slides presented by Slater are almost the same as 

 those of Wainio and ours (published in A'a/M/r,Lo«t/. 182, 1306(1958)). The cytochrome 

 oxidase activity of our cytochrome a preparation is, as I showed, essentially the 

 same as that of the particulate preparation, if the cytochrome a is supplemented 

 with cytochrome c. Therefore, if cytochrome 03 is the true cytochrome oxidase, our 

 cytochrome a preparation should contain it to show the oxidase activity with no 

 cytochrome c. 



With respect to different kinds of cytochrome a present in the preparation which I 

 suppose, and which is supported by Morrison's electrophoretic separation of active 

 from inactive cytochrome oxidase, one may consider that the cytochrome a prepara- 

 tions contained varying amounts of admixed modified cytochrome a, or cytochrome a 

 masked by non-ionic detergent. I believe that our preparation does not contain 

 cytochromes other than cytochrome a and that cytochromes a and c show the oxidase 

 activity but not cytochrome a alone. 



I think that it would be of advantage to consider Pseudomonas cytochrome oxidase 

 and the cytochrome oxidase activity of the cytochrome a, both being double-headed 

 enzymes, at least in their functional state. 

 Slater: Minnaert {Biochim. biophys. Acta 35, 282, 1959) in our laboratory confirmed the 

 observation of Okunuki and his colleagues that the absorption spectrum of cytochrome 

 oxidase preparations in the presence of air and absence of hydrogen or electron 

 donors is not the same as that obtained with K3Fe(CN)6. The difference is in fact 

 somewhat greater than that found by Okunuki, and calculations show that it is not 

 due to the presence of an inactive cytochrome a or a^ which is oxidizable by 

 K3Fe(CN)6, but not by oxygen. 



The same spectrum was obtained whether the K3Fe(CN)6 was added in the presence 

 of air or anaerobically to a preparation reduced spontaneously. 



The positions of the absorption peaks are 



It is thought likely that the spectra obtained with K3Fe(CN)6 and Na2S204 are 

 those of cytochromes a+++ + 03+++, and of a++ + «3^^, respectively. In air, the 

 spectrum is that of a+++ plus a second form of oxidized cytochrome a^, possibly a^ 

 ferry 1. 



It is not known whether both forms of oxidized cytochrome a^ are involved in the 

 enzymic reaction. Both are, however, equally reactive in a system containing ascorbic 

 acid and cytochrome c. 

 Chance: With reference to Morton's point, a number of cogent arguments have been 

 advanced by Keilin, by Slater, and by myself on the separate identities of cytochromes 

 a and 03 (for a summary, see Chance, B., Conference on Oxidative Metal Enzymes, 

 Tokyo, 1957). Perhaps the most easily expressed is that, with intact cells and oxidase 

 preparations, a sudden addition of oxygen to the reduced oxidase causes a greater 

 disappearance of the absorption band at 445 m/< than of that at 605 mn at times of 

 about 10 msec (Chance, Disc. Faraday Soc. 20, 205, 1955). This observation is 

 inconsistent with the hypothesis that these bands represent the a and }' bands of the 

 same haematin group, and the two are thus concluded to belong to different chemical 

 entities. Although we respect the idea that cytochrome oxidase may be one protein 

 molecule (see for example, Ball, Strittmatter and Cooper, J. biol. Chem. 193, 635, 

 1951, who suggest that cytochrome a + 03 be represented as a four-haem protein, 

 and Wainio and Cooperstein, Advanc. Enzymol. 17, 329, 1956), we feel that the 

 experimental results cited above apply equally well under these circumstances. 



To focus the discussion more sharply, we suggest here some speculative configura- 

 tions, not because there are particular data in favour of them, but to evoke discussion 

 and to indicate that basic objections apply to some of the structures. In the first 



