Cytochrome b, 559 



shall try to bring out a few points, which the authors of the papers presented are free 

 to put right if they do not agree. In my view, there are first of all the three groups headed 

 by Morton (Australia), Bocri (Italy) and Nygaard (Norway). The first two of these 

 have no really essential difference in regarding the yeast enzyme as a flavohaemo- 

 protein and, whether they prefer to call it 'cytochrome b^' or 'flavocytochrome 62', 

 these authors are satisfied that they are dealing with a single unit containing proto- 

 haem, flavin and a single enzyme protein, though these compounds can be caused to 

 dissociate under some treatments. There are, however, points of difference between 

 these groups of authors, particularly in regard to the behaviour of the enzyme with 

 different terminal acceptors, and also in regard to substrate specificity. 



Morton and co-workers find that their well-crystallized cytochrome 60 is almost 

 free from non-haem iron and is only slightly autoxidizable while Boeri and Rippa 

 find that their uncrystallized but highly active material is autoxidizable especially at 

 higher oxygen concentrations, but that this autoxidizability is inhibited by excess 

 lactate, probably by binding o active metal (Fe) ions by substrate. Morton and 

 co-workers find that dissociation of part of the flavin increases autoxidizability of the 

 crystalline material, with a decline in activity towards ferricyanide. The best substrate 

 is L-lactate, though oxidation of some other a-hydroxy acids (but not glycerate) 

 occurs, as reported by Morton and co-workers. 



Nygaard is evidently dealing with a very different material, or rather three different 

 non-crystalline materials, prepared from Norwegian yeast. By purification including 

 DEAE-cellulose adsorption, he has obtained these fractions with different activities 

 and different heat-stabilities, all able to oxidize lactate, though with differences in 

 reactivity towards different hydrogen acceptors and in optical specificities. Whereas 

 Morton's preparation contained some deoxyribonucleotide, Nygaard's had poly- 

 ribonucleotides, retained even after dialysis. How much these preparations represent 

 breakdown during extraction is not yet clear. 



The Japanese workers also have made studies of this enzyme. Ogura's kinetic 

 studies establish that the flavin group is involved in the enzymic reaction, supporting 

 the original work of Morton's group. However, Hasegawa and Ogura do not seem 

 to agree entirely with Morton and co-workers on the reaction with ferricyanide. The 

 Japanese work at Osaka demonstrates the alkaline extraction from Japanese yeast of 

 a crystalline haemoprotein having an absorption spectrum resembling the other 

 workers' cytochrome b.^, but devoid of lactate dehydrogenase activity, and having no 

 flavin or non-haem iron. It was also formed by alkaline treatment of a 'yeast lactate 

 dehydrogenase' of the flavohaemoprotein type. Horio and co-workers call this 

 haemoprotein 'cytochrome bo' and suggest that in the intact system a dehydrogenase, 

 containing a bound flavin, is linked to their cytochrome b^, somewhat similarly to the 

 composition of succinate oxidase. The substrate specificity of the partially-purified 

 complex is wider than that observed by Morton and co-workers with their material, 

 and includes glycerate, malate and TPNH. 



While this enzyme system is of a novel and most interesting type, there is one 

 question that I would like to put to the various contributors : what is the metabolic 

 function of L-lactic dehydrogenase in the intact yeast cell? Lactate is not generally 

 considered to be an important metabolite in yeast, although it is known that added 

 lactate can be freely utilized (Meyerhof, Biochem. Z. 162, 43, 1925). There is obviously 

 a problem here. 



My second point concerns substrate specificity. This varies in the different pre- 

 parations studied, and the reason for this interests me very much. With the original 

 preparation of Bach, Dixon and Zerfas, we found (Dickens and Williamson, Nature, 

 Loud. 178, 1118, 1956) that at pH 6-7 glycerate was oxidized to hydroxypyruvate at 

 half the rate of lactate ; only the L-isomer was attacked, and a purified yeast lactate 

 dehydrogenase kindly supplied by Boeri gave a similar result. This raises the possi- 

 bility that an a-hydroxyacid other than lactate might be the natural substrate. 

 . Finally I would like to add a correction to a proposed mechanism for enzymic 

 dehydrogenation of lactate and glycerate to pyruvate and hydroxypyruvate (Dickens 

 and Williamson, Biochem. J. 68, 74, 1958). 



