18 A SYMPOSIUM ON RESPIRATORY ENZYMES 



as cytochromes a, b, and c, are spectroscopically visible in their 

 reduced forms. Their role in biological oxidations so far as we know 



o 



it is shown in Figure 1. As depicted here, oxygen reacts with ferrous 

 cytochrome oxidase, presumably to form an oxygenated compound 

 similar to oxyhemoglobin, as evidenced by the competitive aflBnity 

 shown by carbon monoxide. In the case of cytochrome oxidase, how- 

 ever, the oxygen is able to strike in and oxidize the ferrous iron to 

 ferric. The role of oxygen in biological processes is now ended. 

 Combining with hydrogen ions withdrawn from the acid-base con- 

 tinuum of the cell, it forms water. The ferric cytochrome oxidase thus 

 formed can now bring about the oxidation of the cytochromes a, b, 

 and c. In the presence of cyanide their oxidation is somehow pre- 

 vented. Whether the c)'tochromes react as a chain or individually 

 with ferric cytochrome oxidase we cannot say with certainty. If they 

 react as a chain, we may align them as shown here in view of their 

 relative oxidation-reduction potentials. The reaction involves the 

 transfer of an electron from one iron compound to another without 

 involving oxygen or hydrogen ions in the oxidation. Thus the oxi- 

 dizing agent in the cell that we now have to deal with is ferric 

 iron in organic combination. With what does it react? If we could 

 answer that question, one of the largest gaps in our knowledge of 

 the mechanisms of biological oxidations would be filled. 



Since we can follow the pathway from the oxygen side no further, 

 let us turn our attention to the substrate side, to the studies made 

 upon its activation. The chief tools employed in these studies have 

 been certain dyestuffs capable of undergoing reversible oxidation 

 and reduction. Methylene blue in particular has been widely used; 

 as we now know, its choice was a most fortunate one in view of the 

 relative oxidation-reduction potentials of the systems concerned. By 

 using methylene blue as the oxidizing agent in place of oxygen it 

 was possible to show that the reducing action of various substrates 

 can be elicited only when certain tissue constituents are also present. 

 This was the most striking evidence that had been mustered for the 

 view that substrate activation must take place in biological oxida- 

 tions. In the hands of Thunberg and his co-workers this technique 

 proved most useful in demonstrating the existence of a group of 

 enzymes which were called dehydrogenases or dehydrases because 

 their function appeared to be the activation of the hydrogen of the 

 substrate in preparation for its removal to a suitable acceptor. 



Each substrate or class of substrates, it was demonstrated, pos- 

 sesses its own specific dehydrogenase. Now since leuco-methylene 



