Cytochrome Oxidase Components 339 



pyridine nucleotides in intact mitochondria. Other approaches (sec, for 

 example, Boyer, 1957; Slater, 1958; Wadkins and Lehninger, 1958) have 

 involved studying the relative rates of incorporation of ^-P or ^^O in an effort 

 to evaluate the steps in the phosphorylation process. The phosphorylation 

 steps have been studied by employing uncouplers and antibiotics (Lardy and 

 McMurray, 1959). Slater (1958) has measured the pH optimum of the various 

 steps involved in this process. 



More directly, the classical approach of separation and isolation of the 

 enzymes involved in the phosphorylative steps, has only recently shown signs 

 of being fruitful. The process of oxidative phosphorylation has been con- 

 sidered to be catalysed by a complex of enzymes which are very unstable. 

 Until comparatively recently, the intact mitochondria were the smallest units 

 capable of this process. It is now possible, by various techniques (Green and 

 Crane, 1957; Kielley and Bronk, 1958; Lehninger, Wadkins, Cooper, 

 DevUn and Gamble, 1958) to prepare submitochondrial units which are 

 capable of coupling oxidation and phosphorylation. More recently, it has 

 been possible to subfractionate the complex of enzymes even further and 

 isolate the enzymes involved in the terminal steps in the phosphorylation 

 procedure (Remmert and Lehninger, 1959; Wadkins and Lehninger, 1958). 



Still to be clarified are the enzymic steps in which the electron carriers 

 themselves are involved in the chemical transformations resulting in phos- 

 phorylation. We decided to attack the problem by investigating this 

 phenomenon in the steps involving cytochrome c and cytochromes a and a^. 



It has been clearly demonstrated that in the oxidative sequence from 

 cytochrome c to oxygen, at least one 'high-energy' phosphate can be derived 

 per passage of two electrons (Judah, 1951 ; Maley and Lardy, 1954; Nielsen 

 and Lehninger, 1954). Initial studies in our laboratories demonstrated that 

 when purified isolated cytochrome c was added to the mitochondria, employing 

 ascorbate as the substrate, the rate of both oxidation and phosphorylation 

 increased. The increase in phosphorylation, however, did not parallel the 

 increase in oxidation, and as a result there was a decrease in the P/O ratio 

 with increasing concentrations of added cytochrome c. 



Varying the concentration of mitochrondria in the same type of experiment, 

 showed that the rate of phosphorylation was proportional to the amount of 

 mitochondria at any given concentration of added cytochrome c. This was 

 not true of oxidation. At high levels of mitochondria, the oxygen consump- 

 tion does not increase. 



In the widely used scheme: 



AH^ + B + I-^A^I -[- BH^ 



A and B are general symbols for electron carriers and / is a compound which 

 forms a bond with one of the carriers in which the energy available from elec- 

 tron transfer is conserved ; / is the coupler of oxidation and phosphorylation 



