566 Discussion 



respiratory chain, and more resembles the sluggish reduction of cytochrome b of the 

 non-phosphorylating respiratory chain. 



On Intramolecular Transfer in Cytochrome bg 



Ogura: Although Chance suggested by using rapid spectrophotometric method that the 

 ferricyanide may be reduced through the protohaem of this enzyme, the same result 

 was obtained also in our experiments by the method of overall reaction kinetics. The 

 relationships between Ijv and l/[s] which were obtained in the presence of low concen- 

 trations of ferricyanide were the same as shown in Fig. 4 (B) in the paper of Morton 

 and co-workers (p. 517), namely, these relationships obtained seemed to be parallel to 

 each other. 



Although this result obtained by us does not seem to accord with the data of Morton 

 and co-workers (this volume, p. 518) such discrepancy seems to be caused by the 

 concentration of ferricyanide used. The lower concentrations of ferricyanide were 

 used in our experiments, since the reaction at the initial stage was zero order in respect 

 to the H-acceptor concentration in the presence of 0-5 mM ferricyanide. 

 Morton: The discrepancy between the results obtained by Armstrong and myself, and 

 those of Hasegawa and Ogura, can most probably be attributed to the difference in 

 concentration range of ferricyanide, as suggested by Ogura. Our kinetic studies have 

 to be extended, and we plan to examine the kinetics of the reaction of crystalline 

 cytochrome bo at the lower concentrations of ferricyanide. 



Although Fig. 1 of our paper (this volume, p. 502) shows ferricyanide reacting only 

 with the riboflavin phosphate group, there is reason to believe that ferricyanide may 

 react with either, or both, haem and riboflavin phosphate, according to the conditions 

 of the experiment. This appears to be the situation also in the succinate dehydrogenase 

 system. 



We have been very much interested in the kinetic results obtained with the double- 

 beam instrument since the original report by Chance, Klingenberg and Boeri, Fed. 

 Proc. 15, 231, 1956) of their studies with cytochrome bo. Kinetic studies with this 

 flavohaemoprotein are extremely difficult because of the lability of the material and 

 the results are very much dependent on the conditions of the experiment. In the 

 studies reported here (Morton, Armstrong and Appleby, this volume, p. 501), Arm- 

 strong and I used freshly-crystallized enzyme, kept under anaerobic conditions and 

 in the presence of ethylenediamine tetra-acetate (EDTA). Crystallization is a useful 

 tool in that it enables one to obtain high concentrations of enzyme relatively free of 

 other materials. The material was non-fluorescent, indicating that both the flavin and 

 the haem groups were intact on the protein. 



Whenever such material is oxidized, it rapidly shows some fluorescence indicating 

 some modification (denaturation) of the cytochrome bo. For this reason we have 

 found it very difficult to carry out reliable experiments with oxidized cytochrome bo. 



With reference now to the experiment reported here by Chance (p. 565), from our 

 experience it would be essential (a) to exclude oxygen completely from the system; 

 (b) carry out the experiment in the presence of 10"* m EDTA (or similar metal- 

 binding agent); and (c) use only native non-fluorescent cytochrome bo. Not only does 

 the presence of oxygen increase the rate of appearance of fluorescence, but also the 

 rate of reaction of the cytochrome bo with oxygen is dependent on the degree of 

 denaturation. As already noted (see Morton et al., this volume, p. 501 ; Horio, 

 Yamashita, Yamanaka, Nozaki and Okunuki; this volume, p. 552), the flavin- 

 deficient haemoprotein is readily autoxidizable whereas the intact enzyme is only very 

 slightly autoxidizable. Moreover, the rate of autoxidation of the intact enzyme is 

 dependent on the concentration of lactate (Boeri and Rippa, this volume, p. 524; 

 Morton et al., this volume, p. 513). Clearly the amount of change at 424 m// recorded 

 by Chance (this volume, p. 565) would be dependent on the amount of oxygen 

 present in the system. 



Secondly, if the material used was not completely intact then the portion of intact 

 native protein would react rather sluggishly with the haematin of the portion of 

 denatured material. In this case, one would find only a slow rate of haematin reduction 



