Cytucliroinc bo 571 



indicates a low molecular weight (Montague and Morton, Nature, Loud. 187, 916, 

 1960). 



EFFECT OF OXYGEN ON REDUCED CYTOCHROME b^ 



In the early experiments, oxygen was not rigorously excluded during the filling of 

 the ultracentrifuge cell. In some cases it was observed that the material showed a yellow 

 fluorescence and the solution, when examined with white light, appeared to have a 

 brownish tinge when compared with freshly prepared cytochrome ^g. In the ultra- 

 centrifuge, two additional small boundaries appeared, leading the main boundary. 

 For one such preparation, values of .Vao.w of 7-88, 11-22 and 14-54 x 10"^^ sec were 

 obtained. In addition, the solution to the centripetal side of the boundary appeared 

 to be yellow. 



It is evident that exposure of the enzyme to oxygen may cause (1) dissociation of 

 some of the flavin, which becomes oxidized and therefore fluorescent, accompanied by 

 (2) aggregation (polymerization) of some of the protein, presumably the material from 

 which flavin has dissociated. 



EFFECT OF TREATMENT OF CYTOCHROME Z>2 WITH 

 /7-CHLOROMERCURIPHENYLSULPHONATE (PCMS) 



Previous studies (Appleby and Morton, Nature, Lond. 173, 749, 1954; Boeri and 

 co-workers. Arch. Biochem. Biophys. 60, 463, 1956) have shown that mercurials such as 

 PCMS are powerful inhibitors of the lactate-ferricyanide and lactate-ferricytochrome c 

 reductase activities of cytochrome b^. On anaerobic incubation for about 15 min 

 of intact, non-fluorescent cytochrome b^ in the standard buffer with PCMS at a 

 final concentration of 1 x 10~* m, the solution showed an intense yellow fluorescence, 

 while the colour changed from coral pink to a brownish pink. Spectroscopically the 

 a- and /S-bands of the reduced haemoprotein component appeared unaltered. 



The material so treated sedimented in the ultracentrifuge as a single component, 

 leaving a yellow non-sedimenting component behind. On exposure of the PCMS- 

 treated material to air, the a- and /3-bands of the reduced haemoprotein rapidly 

 disappeared, showing that it was now readily autoxidizable. The oxidized material 

 showed the same sedimentation behaviour as observed before oxidation. The 

 sedimentation coeflficient was not greatly altered from that of intact cytochrome b^. 



It is apparent that treatment with PCMS displaces the flavin prosthetic group, and 

 at the same time prevents the aggregation which occurs in the presence of oxygen. 



As already pointed out (Appleby and Morton, Biochem. J. 73, 539, 1959), at 

 alkaline or acid pH the flavin group is rapidly displaced with corresponding loss of 

 enzymic acitivity. The reaction is catalysed greatly by copper ions (Morton et al., 

 this volume, p. 507), and the rate of autoxidation of cytochrome ^2 is strongly in- 

 fluenced by the ionic strength of the medium (Boeri and Rippa, this volume, p. 524). 



Taken together with the results reported here, these observations suggest that the 

 flavin group may be held by labile hydrogen bonds to thiol groups of cysteine residues 

 of the protein. The reactivity of such thiol groups would expect to be greater at alkaline 

 (> pH 8) as compared with neutral pH values. After displacement of the flavin, the 

 thiol groups may be oxidized to — S — S^ groups, thus forming intermolecular cross- 

 links. When blocked by suitable compounds, the thiol groups are no longer capable 

 of such reactions. This structure may explain the considerable lability of cytochrome 

 62, and shows how modification of the native material may readily occur. There are 

 eighteen ^-cystine groups/haem in cytochrome 62 (see Morton, 1958, loc. cit. ; Appleby, 

 Morton and Simmonds, Biochem. J. 75, 72, 1960). 



The results reported here give further evidence that cytochrome Z>2 is a single protein 

 having flavin and haem prosthetic groups. 



. Although it is possible that the displacement of flavin and the observed aggregation 

 are not directly related, the probable explanation of the observations is that the dis- 

 placement of the flavin uncovers groups which are: (a) reactive with PCMS, (b) 



