Cytochrome b in the Respiratory Chain 



581 



at 448 m/j. This did not occur, unless inliibitors of the chain between suc- 

 cinate and cytochrome b (e.g. malonate) were added (cf. Holton, 1955). 

 A comparison of the traces of cytochromes b and ^3 in Fig. 2 showed that 

 reduction of cytochrome b began about 0-25 sec after the beginning of 



Fig. 2. Reduction of cytochromes h and a^ in heart-muscle preparation by 

 succinate or DPNH from the aerobic steady state to anaerobic steady state. 

 Changes of absorbancy were calculated from the traces obtained at 432 m/x 

 and 448 m/x with Holton's (1957) recorder. The position of the curves on the 

 ordinate is arbitrary. Light path, 0-5 cm. Substrate (5 mM succinate or 1-5 mM 

 DPNH) added at zero time to a suspension of NaNOg-treated heart-muscle 

 preparation (4-35 mg protein/ml) in 004 m phosphate, pH 7-4, 0001 m ethylene- 

 diaminetetraacetate. The Qoj (/"l- Oj/mg protein/hr) at 25° of the preparation 

 was 368 for DPNH and 351 for succinate. 



reduction of cytochrome a^. This sequence would be expected if cytochrome 

 b acted nearer substrate than cytochrome a^. 



In Fig. 3, the reaction rates at different points of the absorbancy curve, 

 divided by the difference in absorbancy between each point and the maximum, 

 have been plotted against time. This quantity d/i/dr . 1/A^ has a formal 

 relationship to a first-order reaction constant, with dimensions sec~^, and 

 has been designated k'^ and k'^^ for the two cytochromes. The A:'s are not 

 constant, but increase from zero during the aerobic steady state to a maxi- 

 mum during the initial stages of the reduction, and decrease sharply again 

 towards the end of the reaction. Although the actual values have no real 

 meaning, being the result of measuring the difference between a reduction and 

 an oxidation reaction, it appears significant that, both with succinate and 

 with DPNH as substrate, k'^ and k'^^ follow similar curves during the final 

 stages of the reduction. We would not expect this behaviour if cytochrome b 



