502 R. K. Morton, J. McD. Armstrong and C. A. Appleby 



and Morton, 1959a). A yield of 10-20 rag of crystals/kg of dried yeast is 

 consistently obtained and, with only minor modifications, the procedure as 

 described in detail by Appleby and Morton (1959a) has been successfully 

 used for isolation of crystalline cytochrome b.^ from a number of different 

 strains of commercial bakers' yeast obtained in Australia and from other 

 countries. Boeri (personal communication) has recently reported isolation 

 of crystalline cytochrome b^ from Italian yeast by this procedure. Because 

 of the ready dissociation of flavin from this cytochrome (see later), it is 

 essential to observe the precautions indicated by Appleby and Morton 

 (1959a) in order to avoid modification of the cytochrome during isolation. 



oxygen Ferricyanide methylene blue 



2:6-dichlorophenol indophenol 



Riboflavin >- protohoem >- cytochrome Q 



phosphate 



oxygen 



c ytochrome bj 



Fig. 1. Scheme for interaction of substrate and acceptors with cytochrome 62 

 (Morton, 1955a; Appleby and Morton, 1954, 1955). 



Crystalline cytochrome b^ catalyses the reduction by lactate of ferricyanide, 

 cytochrome r, methylene blue, 2 : 6-dichlorophenol indophenol and oxygen 

 (Appleby and Morton, 1954, 1959b; Armstong and Morton, 1959). Based 

 on detailed chemical and preliminary kinetic studies, it was proposed (Morton, 

 1955; Appleby and Morton, 1954, 1955) that various hydrogen (or electron) 

 acceptors interact with cytochrome b^ as shown in Fig. 1. This scheme 

 postulates that reduction of cytochrome c and of certain dyes involves an 

 intramolecular hydrogen (or electron) transfer between the flavin and haem 

 groups attached to the one protein molecule, whereas in other systems such as 

 the heart-muscle succinate-cytochrome c reductase, transfer takes place 

 between the flavin and haem groups of different proteins, succinate dehydro- 

 genase (flavoprotein) and cytochrome b (haemoprotein) respectively (Morton, 

 1955). The chemical and kinetic evidence presented here further establishes 

 that cytochrome b^ is a flavohaemoprotein and shows that reduction of 

 cytochrome c, ferricyanide and dyes involves different mechanisms. 



CHEMICAL PROPERTIES OF CRYSTALLINE CYTOCHROME b. 

 AND OF DERIVED PROTEINS 



Prosthetic Groups 



Maximum lactate dehydrogenase activity (approx. 7750 /tmoles of ferri- 

 cyanide or 5870 //moles of cytochrome c reduced/hr/mg of enzyme at 20°C) 



