504 R. K. Morion, J. McD. Armstrong and C. A. Appleby 



is found in twice-recrystallized cytochrome b^, the activity is constant after 

 further recrystaUizations (Appleby and Morton, 1959a, b). Chemical 

 analyses of such material are shown in Table 1 . 



The absorption spectrum of the pyridine haemochrome of cytochrome 63 

 (Bach et al., 1942; Appleby and Morton, 1954, 1959b) and the properties 

 of the isolated haemin (Armstrong and Morton, 1959) show that the haem 

 group is identical with protohaem. The identity of the flavin group with 

 riboflavin phosphate (FMN) was established by paper chromatography and 

 by fluorimetry (Appleby and Morton, 1954, 1959b). The amounts of proto- 

 haem (0-77%), riboflavin phosphate (0-55%) and iron (0-077%) all indicate 

 a minimum weight/mole of approx. 80,000 g, with equimolecular proportions 

 of flavin and haem and the absence of non-haem iron. 



Mahler and Green (1954) and Mahler (1956) have proposed that the inter- 

 action of flavoproteins with cytochrome c involves an essential flavin-bound 

 metal (iron, copper, molybdenum or related metals). Green and Beinert 

 (1955) predicted that cytochrome b.^ would contain a non-haem metal group 

 because it reacted with cytochrome c and with ferricyanide. However, any 

 mechanism of cytochrome Z72~cytochrome c interaction involving such 

 metal groups is not supported by the analyses of crystalline enzyme (Table 

 1 and Appleby and Morton, 1959b). Morton (1955a) had pointed out that 

 non-haem iron, copper, cobalt, nickel, silver and related metals are either 

 absent or occur only in trace amounts in crystalhne cytochrome 62. Moreover, 

 addition of ferrous sulphate to crystalline cytochrome b^ causes no stimu- 

 lation of the activity with cytochrome c (Appleby and Morton, 1959b) and 

 dialysis of the non-crystalline cytochrome b^ of Boeri et al. (1955) against 

 chelating agents (o-phenanthroline, 8-hydroxyquinoline, or N,N'-dihydroxy- 

 ethylglycine) causes no loss of enzymic activity (Boeri and Tosi, 1956). This 

 evidence establishes that the interaction of cytochrome Z?2 with cytochrome c 

 (or other acceptors) cannot involve metals other than the iron of the proto- 

 haem prosthetic group and precludes mechanisms of the kind postulated by 

 Green and Beinert (1955) and by Mahler (1956). It should be noted, however, 

 that Boeri and Tosi (1956) found 8 g atoms and Boeri and Cutolo (1958) 

 1 g atom of non-haem iron/mole of haem in non-crystalline cytochrome b^ 

 and Boeri and Tosi (1956) have proposed that these iron atoms have an 

 essential function in the enzymic reduction of cytochrome c. On the basis of 

 the findings of Boeri and colleagues, Mahler (1956, p. 224) has described 

 yeast lactate dehydrogenase as a metalloflavoprotein. However, the evidence 

 strongly suggests that the non-haem iron found by Boeri and co-workers is 

 an impurity in their preparations of cytochrome bo. 



Crystalline cytochrome b,, sediments at pH 6-8 and at 0°C as a single 

 component (sedimentation constant, -S^yw 8-485'). The enzyme may 

 existasa dimerwith two moles each of riboflavin phosphate and of protohaem/ 

 mole of enzyme. Prolonged electrophoresis at pH 6-6, 7-05 and 8-8 also 



