METHODS OF LOCALIZATION 517 



electrons at any point in the sequence will modify the steady-state spectral 

 characteristics. Under favorable circumstances these changes may be ob- 

 served in preparations of living cells. Assuming a generalized electron- 

 transport sequence such as: 



SHj -> DPX+ -> FAD -^ cyt-b -> cyt-Cj ^* cyt-c -> cyt-a -> oxidase -> O2 



where the arrows indicate the direction of electron flow, the inhibition of a 

 particular step will slow down or prevent reduction of the components to 

 the right of the block and oxidation of the components to the left of the 

 block. This principle was first applied by Keilin in 1925 in his classic paper 

 on the nature of the cytochromes and extended to more accurate localiza- 

 tions in later reports. More recently this technique has been refined and 

 applied extensively to many systems by Chance (1958; Chance and Wil- 

 liams, 1956; Chance and Hess, 1959). 



The typical simple four-banded spectrum of the reduced cytochromes was 

 observed in many tissues by Keilin (1925) and the disappearance of these 

 bands was noted upon oxidation. A yeast suspension under anaerobic con- 

 ditions thus exhibited the bands of the reduced cytochromes; upon shaking 

 with oxygen these bands faded and disappeared. However, if cyanide 

 were present, the reduced spectrum remained when the cells were exposed 

 to oxygen. It was also found that when cyanide was added to an oxygenated 

 suspension, the bands of the reduced cytochromes appeared rapidly. It 

 was concluded that cyanide acted on some component between oxygen and 

 the cytochromes; 3 years later this was identified with the terminal oxidase 

 (called indophenol oxidase at that time and now known as cytochrome 

 oxidase) (Keilin, 1928). Urethane, on the other hand, inhibited respiration 

 in a different manner because it was shown to block the reduction of the 

 cytochromes without having any effect on their oxidation (Keilin, 1925). 

 A yeast suspension treated with urethane and shaken with oxygen showed 

 the cytochromes to be oxidized; if cyanide was then added, the cytochromes 

 did not become reduced as they would have been in the absence of urethane. 

 Urethane must therefore act on some step between the substrate and the 

 cytochromes. Keilin was able to propose a tentative sequence: 



(urethane) (cyanide) 

 Substrate > cytochromes > Oj 



An approximate localization for the sites of inhibition could be made be- 

 fore the actual components of the electron transport system were known 

 and the subsequent elucidation of the components has been aided by the 

 use of these and other inhibitors. 



The later analysis of the site of action of cyanide (Keilin and Hartree, 

 1939) illustrates another but more direct technique. When the compo- 

 nent with which the inhibitor reacts possesses a characteristic spectrum. 



