Properties of Cytochrome b,^ 507 



catalyse autoxidation. The inactivating effects of copper salts, and the 

 protection by EDTA, is shown by Table 3. 



Both the metal-catalysed and the direct autoxidations cause appearance of 

 fluorescence due to dissociation of the FMN group of cytochrome ^2 (Appleby 

 and Morton, 1959b). Since copper salts readily catalyse oxidation of many 

 thiols (see Barron, 1951), the probable involvement of thiol groups is 

 indicated. 



In the presence of substrate, /?-chloromercuribenzoate (10"^ to 10"* m) 

 strongly inhibits lactate dehydrogenase activity (Appleby and Morton, 1954; 

 Boeri et al., 1955) whereas mono-iodoacetate (10^^ m) under similar condi- 

 tions has little effect (Boeri et al., 1955). Hence some thiol groups of the 

 enzyme are protected from alkylation, although readily reacting with the 

 powerful mercaptide-forming reagent. Inhibition by /?-chloromercuribenzo- 

 ate causes appearance of strong fluorescence, indicating displacement of the 

 riboflavin phosphate group by this reagent (Armstrong, Coates and Morton, 

 1960). 



Since the active enzyme itself is not fluorescent, the imino group of the 

 iso-alloxazine ring is probably bonded to a group of the protein, and it seems 

 likely that there is strong hydrogen-bond formation between the imino group 

 and an ionised thiol group of the protein. There are 18 S-containing residues/ 

 mole of haem (Table 1). Many of these may occur as cystine, and relatively 

 few as cysteine. Flavin fluorescence also appears under acid ( < pH 4) and 

 alkahne (> pH 9) conditions, under which conditions activity with ferri- 

 cyanide and with cytochrome c is greatly decreased (Appleby, 1957; Appleby 

 and Morton, 1959b). 



The enzyme is considerably protected by lactate, and, at the same time, 

 there is marked reduction in appearance of flavin fluorescence. The ready 

 appearance of flavin fluorescence in the oxidized enzyme, and protection by 

 lactate and EDTA, suggest that the dissociation constant of the flavin group 

 of oxidized cytochrome 02 is very much greater than in the reduced cyto- 

 chrome. The dissociation constant of the oxidized form of the 'old yellow 

 enzyme' is over 100 times greater than that of the reduced form (Nygaard and 

 Theorefl, 1955; Vesthng, 1955). If the flavin group is bound by thiol groups 

 as proposed, oxidation of thiol groups after dissociation of the flavin would 

 prevent recombination of the flavin and haemoprotein. The dissociation of 

 flavin from cytochrome b^ is therefore strictly reversible only under conditions 

 which prevent oxidation of the thiol groups of the apoproteins. The substrate 

 probably protects enzymic activity by maintaining the FMN group in the 

 reduced state and thus lowering the dissociation from the apoprotein. 



The Haem-ProteUi Link 



As In other cytochromes, the haem group is strongly bound by the apo- 

 protein, but may be split by treatment with acid-acetone (Armstrong and 



