574 Discussion 



depending on the degree of denaluration and in this case too, the rate of autoxidation 

 of each individual sample will be a function of the ionic strength of the medium. 



WiNFiELD : In regard to the question on rate of oxygen uptake by the enzyme, it seems quite 

 possible that a small change in the mode of attachment of flavin to the protein, during 

 isolation of the enzyme, could result in the flavin becoming autoxidizable, as indicated 

 by Morton ; vice versa, the capacity of the flavin moiety to combine with oxygen, if 

 it is present in the native enzyme, could be lost during isolation. 



Williams: The autoxidation of cytochrome b^ is written by Morton and co-workers as an 

 attack on the haem iron. The results of Boeri and colleagues indicate that the reaction 

 is an attack on free iron. The experiments of Boeri and co-authors are very like those 

 of Warburg on the catalysed oxidation of cysteine by haemochromes. Warburg 

 takes the point of view of Morton. In a discussion of this reaction I have proposed 

 that Warburg's mechanism is incorrect (Williams, Chem. Rev. 56, 299, 1956), and I 

 have cited evidence in favour of an attack essentially the same as Boeri's discussion 

 of his reaction. Could Morton and Boeri comment on the diff"erences in their 

 observations which lead them to write the reaction differently ? 



Morton: Oxidation of cytochrome ^2 apparently proceeds in several stages. The intact 

 flavohaemoprotein has a very low activity with oxygen. We are in agreement with 

 Boeri and his colleagues that water is the product of this reaction. This may involve 

 a reaction of oxygen with the haem iron. Alternatively if the flavin of the intact 

 reduced cytochrome exists as FMNH in 'Protein-FMNH-haem-Fe+++' in oxidized 

 cytochrome b.^, (see Boeri and Rippa, this volume, p. 524), an attack on the flavin is 

 possible. Whatever the mechanism, some flavin prosthetic group becomes fluorescent, 

 probably due to oxidation of thiol groups as suggested earlier (Morton, this volume, 

 p. 571 ). The riboflavin phosphate which is no longer bonded to the protein as in native 

 cytochrome 62 possibly due to a bond between the flavin and haem having been broken, 

 may now react directly with oxygen to form HoOo. Evidence for the formation of 

 HoOa during prolonged exposure to oxygen is provided by the protection obtained 

 with catalase (see Morton and co-workers, this volume, p. 506). H2O2 may rapidly 

 oxidize the labile thiol groups, causing further denaturation of the flavohaemoprotein, 

 and dissociation of flavin. 



Denatured cytochrome b.^ formed by disruption of the normal bonds between the 

 flavin prosthetic groups and the protein (as, for example, by treatment of cytochrome 

 Z)o with />-chloromercuriphenylsulphonate), is highly autoxidizable. 



It would appear that the presence of the flavin group hinders attack of oxygen on 

 the haem iron of intact cytochrome b.,. This may be due to the fact that the flavin is 

 bonded directly to the haem iron. Alternatively, the presence of the flavin group is 

 associated with folding of the polypeptide chains which restricts oxygen attack on the 

 haem group; displacement of the flavin causes unfolding of the polypeptide chains 

 and facilitates oxygen reaction with the haem. I consider that the reaction of denatured 

 cytochrome b.. with oxygen is probably analogous to the reaction of the denatured 

 cytochrome c (at pH > 12) with oxygen. 



Winfield: Is it known as to how the amount of cytochrome b^, in yeast is affected by the 

 oxygen tension during growth ? 



Boeri: The cytochrome Zjj is particularly abundant when the yeast is well aerated and in 

 stationary conditions of growth. Anaerobic yeast lacks cytochrome b.^,, but has instead 

 other enzymes acting on lactate. 



