Cytochrome b in the Respiratory Chain 593 



into account raises his calculation of the potential from —40 mV to +32 mV at pH 7-4. 

 Assuming that the potential of cytochrome b itself is the same at pH 70 as at pH 7-4 

 and allowing for the fact that at pH 7-0 the potential of the succinate/fumarate system 

 is some 24 mV more positive than at pH 7-4, Ball's data correspond to a potential of 

 +56 mV at pH 7-0. Finally it may be noted that Holton and Colpa-Boonstra's calcu- 

 lations are based on the data of Borsook and Schott (/. biol. Chem. 92, 535, 1931) for 

 the potential of the succinate/fumarate system (+24 mV at pH 70), while Ball used a 

 value corresponding to +4 mV at pH 70. Thus, calculated on the same basis and 

 allowing for the endpoint error in Ball's work, his observations correspond to a value 

 of about +76 mV at pH 7-0, in very close agreement with the estimate of +77 mV 

 made by Holton and Colpa-Boonstra. Chance's data also give practically the same 

 value. Thus, there is no discrepancy between three sets of data. The difference lies in 

 interpretation. There does appear to be some discrepancy with Hill's data, but dis- 

 cussion of possible reasons must await pubHcation of details of his measurements. 

 (See Holton and Colpa-Boonstra {Biochem. J. 76, 179, I960).) 



On the Redox Potential of Cytochrome b, the Kinetics of Reduction of 

 Cytochrome b, and the Existence of Slater's Factor 



Chance: I am quite in sympathy with the approach of Holton and Colpa-Boonstra, to the 

 question of how to interpret the succinate-fumarate titration of cytochrome b of the 

 Keilin and Hartree heart-muscle preparation and have been actively considering this 

 ever since we found that a dithionite-reducible pigment would cause errors in the redox 

 potential of cytochrome b (Chance, Nature, Lond. 169, 215, 1952). In fact the chief 

 reason for a delay in publishing the results on cytochrome b titrations was that the 

 kinetic and equilibrium data were inconsistent. I have refrained from calculating an 

 oxidation-reduction potential, since, regardless of admonitions to the contrary, it 

 would ultimately appear in various tables as a firmly estabhshed value. The data which 

 have caused me to hesitate to derive the thermodynamic quantity are: 



(1) The failure to obtam a reduction of cytochrome b by redox couples of higher 

 potential. The 100 mV figure for the antimycin-A treated material (Slater and 

 Colpa-Boonstra, this volume, p. 584) suggests that ascorbate would be effiective. 



(2) The ratio of the kinetic constants do not equal the 'equilibrium' constant. There 

 is the possibility of a simulated equilibrium due to a fumarate reductase or 

 similar activity. In this case, a variable cytochrome b+++lb++ ratio could be 

 obtained by variation of the fumarate/succinate ratio. It is probable that a fairly 

 sophisticated reaction sequence would have to be formulated to fit exactly the 

 experimental data (Takamiya, /. Biochem. Tokyo 46, 1037, 1959). 



(3) HUl's ferric-ferrous oxalate titrations presumably involve a direct reaction with 

 cytochrome b, and are therefore scarcely to be ignored. The 75 mV discrepancy 

 between his value and that which can be calculated from my data is surely beyond 

 any measurement error. The procedure used in Hill's study has been described 

 in detail (Hill, R. In Modern Methods of Plant Analysis, Ed. Paech and Tracy, 1, 

 401, 1956; Bendall and Hill, New Phytol. 55, 206, 1956). It would appear that 

 any objection to Hill's results on cytochrome b would also apply to his results on 

 cytochrome b.,. 



A second general point is the question of whether cyanide specifically slows the 

 reduction of cytochrome b. It should be noted that we have used cyanide to measure 

 the kinetics of reduction of cytochrome b of liver mitochondria, and that the rate is 

 rapid. This point can readily be tested by using azide, hydrogen sulphide, or carbon 

 monoxide instead of cyanide: has Colpa-Boonstra tested these to determine whether 

 the reduction of cytochrome b on adding succinate is faster in the absence than in the 

 presence of cyanide? 



The suggestion that cyanide is an inhibitor of cytochrome b reduction would allow 

 an even more critical test of the idea of a cytochrome b 'by-pass': it is observed that 

 the rate of reduction of cytochrome c is rapid compared with that of cytochrome b in 



