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



The results of Yamashita et al. (1958) show that the flavin-free haemo- 

 protein derivative of cytochrome b^, is reduced by active cytochrome b^ (yeast 

 lactate dehydrogenase) at only about 0-02 times the rate of cytochrome c. 

 Assuming that the haemoprotein derivative of cytochrome b^ is undenatured, 

 it is clear that an inter-molecular reaction between flavoprotein and this 

 haemoprotein could not account for the observed rate of reduction of cyto- 

 chrome c. 



Kinetic studies with DCIP as acceptor are being carried out but only 

 preliminary results are available as yet. However, comparison with the 

 succinate dehydrogenase system, as discussed by Morton (1955a) suggests that 

 dyes interact with cytochrome b.^ at the haem, rather than at the flavin group, 

 as shown in Fig. 1. Recent results of Horio, Yamashita and Okunuki (1959) 

 are consistent with this formulation. 



SUMMARY 



1. The results presented here show that cytochrome b^ is a flavohaerao- 

 protein having equimolecular proportions of riboflavin phosphate and of 

 protohaem and having lactate dehydrogenase activity. A deoxyribose poly- 

 nucleotide associated with the crystalline enzyme has no influence on this 

 enzymic activity. Cytochrome b^ contains no iron in excess of that in the 

 protohaem group. 



2. The weight/mole of haem is 80,000 g. 



3. There is considerable specificity of cytochrome b., at the substrate- 

 binding site. 



4. The linkage between flavin and protein appears to involve hydrogen 

 bonding of the imino group of the isoalloxazine ring to a cysteine residue of 

 the protein. 



5. The amino acid composition of cytochrome b^ is given. Ligands at 

 positions 5 and 6 of the iron atom of the protohaem arc provided by amino 

 acid residues of the protein; probably one position at least is occupied by an 

 imidazole group of a histidine residue. 



6. An intermediate oxidation state of the cytochrome has been detected 

 spectroscopically. The shift of the absorption bands to longer wavelengths as 

 compared with those of the fully reduced cytochrome suggests interaction 

 between substrate, flavin and haem groups. 



7. The change in autoxidizabiUty of cytochrome b^ on dissociation of the 

 flavin, and other evidence, indicate a close juxtaposition of the flavin and 

 haem groups of the protein. 



8. Kinetic evidence supports different mechanisms for the reactions of the 

 enzyme with ferricyanide and with cytochrome c. The mechanism of the 

 reaction of cytochrome b^ with various acceptors is discussed. 



9. The results may be of significance for understanding hydrogen 

 transport during terminal respiration in mitochondria. 



