Properties of Cytochrome b^ 



519 



The enzyme-lactate complex is one in which the greatest part of the two 

 electrons of the lactate are donated to the flavin moiety. The oxidation of 

 this complex by ferricyanide takes place through a ternary complex. This is 

 consistent with the general behaviour observed for flavoprotein enzymes. 



The reaction with cytochrome c requires an intermediate reduced form of 

 the enzyme. It is postulated that the protohaem moiety of cytochrome b^ 

 reacts at the same site as the ferricyanide, oxidizing the lactatc-flavin complex, 



Fig. 6. Lactate-cytochrome c reductase activity about 2 x 10"^ m crystalline 

 cytochrome b^ in 0-03 m sodium pyrophosphate-HCl buflFer at pH 8-0 and 30°C. 

 Heart-muscle cytochrome c purified by resin chromatography was used. 



Units are as follows: v and K„ AE550 m///min ; [S], L(+)-lactate, mmoles/1.; 

 [A].r, heart-muscle cytochrome c, mmoles/1. 



Plot I. Curves for 1 5 /m cytochrome c ( — O — ) and 82 /<m cytochrome c 

 (— n— ). Compare Fig. 4, Plots A and B. 



Plot. II. Curves for IjK,,,^ (— D— ) and for 1/K, (— O— ). 



and thus forms the intermediate reduced form of the enzyme. This is in turn 

 oxidized by the cytochrome c. 



These observations raise the question as to whether the reactions between 

 the flavin and haem groups of the yeast lactate dehydrogenase system are 

 truly intramolecular, viz. between two prosthetic groups of the one enzyme, 

 or intermolecular, viz. between prosthetic groups of two different proteins. 

 The former view is supported by the extensive purification studies, physical 

 studies, and degradation studies of the enzyme as discussed in the foregoing 

 section. Moreover, Chance, Klingenberg and Boeri (1956) titrated cyto- 

 chrome h.^ with lactate and observed that the reduction of the flavin and haem 

 portions is virtually simultaneous. This implies a close juxtaposition of the 

 flavin and haem groups. 



