518 11. LOCALIZATION OF THE SITE OF INHIBITION 



this spectrum of absorption is usually altered by reaction with the inhibitor 

 and this constitutes positive evidence for the locus of inhibition. It was 

 possible to demonstrate the absorption bands of another component of 

 the cytochrome system and this was termed cytochrome a^. Upon reaction 

 with cyanide or carbon monoxide, these bands changed in a manner in- 

 dicative of an inhibitor complex, and it was thus possible to equate cyto- 

 chrome ttg with the terminal oxidase and the exact site of action of these 

 inhibitors. 



The power and accuracy of these methods have been markedly improved 

 with the development of rapid difference spectroscopy by Chance. It is 

 now possible to determine precisely the oxidation-reduction state of each 

 component of the electron transport sequence under a variety of conditions. 

 During activity in the respiratory chain, it is now known that every com- 

 ponent exists partly in the oxidized and partly in the reduced state, the 

 fractions depending on the relative activities of the enzymes in the se- 

 quence and the supply of substrates, oxygen, and phosphate acceptors 

 (Chance and Williams, 1956). Let us illustrate the behavior of such a system 

 during inhibition by outlining the results with antimycin. When antimy- 

 cin was added to a mitochondrial suspension in a steady state of activity, 

 the cytochromes a^, a, and c became completely oxidized, while cytochrome 

 b and the nucleotides became completely reduced (Chance and Williams, 

 1956). The site in the chain where reduction changes over to oxidation is 

 called the "crossover point" by Chance and it is to this locus that the 

 inhibitory attack is attributed. In this case, the crossover point is between 

 cytochromes b and c. Antimycin could thus act either on an unknown 

 component between these cytochromes or it could react with cytochrome 6 

 to prevent its oxidation; reaction with cytochrome c is unlikely because 

 it can be reduced readily upon addition of ascorbate. It is possible to lo- 

 calize the action more closely inasmuch as antimycin has been shown to 

 block the reduction of cytochrome Cj, which operates between cytochromes 

 b and c (Keilin and Hartree, 1955). 



Spectroscopic determinations may be combined with additions of spe- 

 cific electron donors or acceptors. In the hypothetical transport chain: 



I 

 A^B-i->C^D->E (11-4) 



donor acceptor 



where a block has been established between B and C, the addition of a 

 donor from which C can accept electrons will circumvent the block; this 

 was the case in the addition of ascorbate above. Likewise, addition of an 

 acceptor that can take electrons from B will allow the reaction to proceed. 

 There will be, of course, corresjionding changes in the oxidation-reduction 



