594 Discussion 



cyanide-treated preparations (see Chance, this volume, p. 564). Whether or not this 

 discrepancy is due to cyanide inhibition of cytochrome b reduction, it is clear that 

 electrons can be transferred much more rapidly to cytochrome c than to cytochrome 

 b; i.e. electron transfer to cytochrome c does not need to pass through cytochrome b 

 in the Keilin and Hartree heart muscle preparation.* 



A third point refers to the Fig. 3 of the paper by Slater and Colpa-Boonstra in which 

 the rates of reduction of cytochromes 03 and h in the aerobic-anaerobic transition are 

 compared. 1 would first like to point out that they may have selected from their 

 data an experimental condition which obscures tiie very phenomenon to be considered, 

 namely a measurable time difference in the start of the reduction of cytochromes b and 

 03. Although I feel that they have an effective instrument, it may be that its response 

 time is inadequate to show the prior reduction of cytochrome 03 under the experimental 

 conditions and that the slower kinetics in the presence of malonate are more faithfully 

 recorded. We have often observed in the presence or absence of malonate the pheno- 

 menon which Slater and Colpa-Boonstra observed in tlie presence of malonate. 



Lastly I would be pleased to hear more details of the destruction of cytochrome b' 

 by BAL. How is this related to the tentative identification of the BAL-sensitive factor 

 as a haematin? Does not this experiment remove all data in favour of a haematin 

 'factor". 

 Slater: Any value for the oxidation-reduction potential for cytochrome b must be to 

 some degree uncertain, so long as the pure, native cytochrome is not available. How- 

 ever, it seems to me that determinations of the equilibrium reached with succinate 

 and fumarate in the absence of oxygen are useful. For one thing, Holton and Colpa- 

 Boonstra have shown experimentally for the first time that one molecule of succinate 

 reduces two of cytochrome b. The oxidation-reduction potential is one of the con- 

 ventional methods of expressing the value of the equilibrium constant. 



The 'failure' of ascorbic acid to reduce cytochrome b could be a kinetic 'failure', 

 rather than a thermodynamic difficulty. (As a matter of fact, I have reported that a 

 faint band of cytochrome b appears 30 min after the addition of ascorbate to a heart- 

 muscle preparation in the absence of air (Slater, Biocheni. J. 45, 1, 1949)). 



In the anaerobic steady state which is reached after exhaustion of oxygen by the 

 succinate oxidase system, the only hydrogen donor present in a concentration appreci- 

 ably above that of the catalysts in the enzymic system is succinate, while the only 

 hydrogen acceptor present in these concentrations is fumarate. A fumarate reductase 

 could not operate in the absence of a hydrogen donor. The most that it could do 

 would be to change the concentrations of succinate and fumarate by bringing about 

 the following sort of equilibrium 



succinate -1- E ?^ fumarate -f EHo 

 where E represents fumarate reductase. But since the concentrations of succinate 

 and fumarate present are far above those of any possible enzyme present, this error 

 would be completely negligible. 



We are fully aware of Hill's measurements of the oxidation-reduction potential of 

 cytochrome b which are based on titrations with both ferric/ferrous oxalate, and with 

 fumarate/succinate. Until details of his measurements are published, it is not possible 

 to discuss the reasons for the difTerence, which is perhaps less than it now appears. 



There is no difference between Chance and ourselves concerning the interpretation 

 of the slow reduction of cytochrome b (in comparison with that of cytochromes c or 

 «3) by succinate in the presence of cyanide. Under these conditions, it is clear that 

 cytochrome b is not on the pathway of reduction of cytochrome c by succinate. 

 However, we do differ concerning the interpretation of the other experiments in which 

 the reduction of cytochrome b is followed during the exhaustion from the suspension 



* Added in proof. In an experiment performed after this symposium, it has been found 

 that cytochrome b of heart muscle preparation is reduced at the same rate in 1-7 mM NaCN 

 as in 30 mw NaN3. Tlius, a specific inhibition of cytochrome b reduction is unlikely and 

 the idea that cytochrome b is 'dislocated' in non-phosphorylating preparations appears 

 preferable (Chance, 1958a). 



