606 B, Chance 



It is seen that the rates at which flavoprotein and ubiquinone are reduced 

 are comparable, whereas the rate at which cytochrome b is reduced in the 

 absence of antimycin-A is about 40 times slower than that of the other two 

 components and is, in the presence of antimycin-A approximately six times 

 slower (Chance, 1958a). Similar data were used earlier (Chance, 1952) to 

 demonstrate that cytoclirome b is not a member of the electron transfer 

 chain of non-phosphorylating preparations. Cytoclirome c is reduced 

 somewhat more slowly than flavoprotein for two reasons. First, electron 

 transfer has proceeded farther down the chain, and second, cytochrome a is 

 acting as a potent oxidant for cytochrome c. Even so, the rate of cytochrome 

 c reduction is large compared to that of cytochrome b in the presence or 

 absence of antimycin-A. In the uninhibited preparation oxygen is reduced 

 at a rate of 0-44 /tmoles of Og 1.^ sec.^^ in terms of two equivalents/sec. This 

 value is about twice that measured for ubiquinone, in agreement with the 

 data of Redfearn (1959). It is important to compare, in addition to the 

 slopes of the reaction kinetics, the time for completion of the trace for the 

 275 mfJL component with that of the cytochrome c component ; the reduction 

 of cytochrome c is complete by about the time the reduction of ubiquinone 

 reaches its maximal velocity. 



The Function of Cytochrome c^ in the Steady State 



Although detailed kinetic studies of the cytochrome components dis- 

 tinguishable at room temperature can be made on the intact respiratory 

 chain as described above, the obscuring of cytochrome q by cytochrome c 

 has prevented our obtaining even preliminary information on the partici- 

 pation of cytochrome c^ in electron transfer in the Keilin and Hartree heart- 

 muscle preparation, or indeed in phosphorylating mitochondria. Indirect 

 data on the fragmented system have led Keilin and Hartree (1955) to recom- 

 mend the inclusion of cytoclirome q as a member of the respiratory chain, 

 but its action in the steady state of respiration has never been reported. 



Studies of the low temperature difference spectra of mitochondrial sus- 

 pensions frozen rapidly from the aerobic steady state show remarkably little 

 change in the percentage of oxidation and reduction of the cytochrome 

 components such as c, a and b (Chance and Spencer, 1959). This observation 

 leads to the supposition that the steady state of cytochrome q would be 

 similarly independent of freezing and that the ready delineation of this 

 component at liquid nitrogen temperatures would lead to a reasonable 

 estimate of its room temperature steady-state value. An experiment to this 

 point is recorded in Fig. 3, which represents a frozen steady state of beef 

 heart mitochondria (kindly supplied by Dr. D. E. Green) with succinate as 

 substrate. (See Discussion, this volume, p. 458.) 



It is seen that the a-band of cytochrome q shows a clear reduction in the 

 steady state: the value computed is 67%, which is very close to that of 



