Fioperdes of Cytochrome b^ 505 



indicates that crystalline cytochrome ^2 contains no protein other than the 

 flavohaemoprotein (Appleby and Morton, 1960). The amino acid com- 

 position (Table 1) is rather similar to that of pig-heart diphosphopyridine 

 nucleotide (DPN)-diaphorase (a flavoprotein) and to that of cytochrome c 

 (a haemoprotein) (Morton, 1958; Appleby, Morton and Simmonds, 1960). 

 The only unique feature is the apparent absence (< 0-3 %) of methionine which 

 usually accounts for 0-5 to 5% of the dry weight of enzymes and other 

 proteins. This feature of the amino acid composition thus supports the 

 purification studies and the physicochemical analyses in showing that only 

 one protein component is present in the crystalline enzyme. 



The preparation from yeast of a crystalline haemoprotein which has an 

 absorption spectrum resembling (between 400 and 600 m//) that of cytochrome 

 b<^ has been reported by Okunuki and colleagues (Yamashita et al., 1957). 

 The preparation has no lactate dehydrogenase activity and contains no 

 flavin group (Yamanaka et al., 1958). This could imply the existence in yeast 

 of two distinct proteins, a flavoprotein lactate dehydrogenase, and a haemo- 

 protein which acts as the specific hydrogen (or electron) acceptor for the 

 flavoprotein dehydrogenase. The homogeneity of crystaUine cytochrome b2 

 of Appleby and Morton (1954, 1959a) having lactate dehydrogenase activity, 

 however, does not support this view. Moreover, it is significant that equi- 

 molecular amounts of flavin and of haem are found in preparations of 

 enzymically-active cytochrome ^2 obtained by such different procedures as 



(a) extraction with butanol-saturated lactate, fractionation with acetone and 

 crystallization at low ionic strength (Appleby and Morton, 1954, 1959a); 



(b) fractionation of a yeast autolysate with ammonium sulphate and treatment 

 with calcium phosphate gel (Boeri et al., 1955); and (c) grinding of yeast 

 cells, fractionation with acetone and chromatography on a diethylamino- 

 ethyl (DEAE)-cellulose column (Nygaard, 1959). Yamashita et al. (1957) 

 used autolysis for 7 days at pH 8-5 followed by fractionation with ammonium 

 sulphate and chromatography on a cation-exchange resin (Duolite CS-101) 

 buffered at pH 5-0. Experience with crystalline cytochrome Z?2 (Appleby, 

 1957; Appleby and Morton, 1959b) strongly suggests that the conditions of 

 prolonged autolysis and of resin-column treatment used by Yamashita et al. 

 (1957) would cause dissociation of the flavin prosthetic group of cytochrome 

 b2 and consequent loss of dehydrogenase activity. It is therefore concluded 

 that the haemoprotein obtained by Yamashita et al. (1957) is a modified 

 protein derived from cytochrome b.^. This conclusion is supported by kinetic 

 studies as presented here. 



The Flavin-Protein Bonds 



Loss of enzymic activity of cytochrome Z>2 is frequently accompanied by the 

 appearance of flavin fluorescence (Appleby and Morton, 1954; Boeri et al., 

 1955); this is prevented, and activity is protected by exclusion of oxygen, by 



