624 Discussion 



oxidative phosphorylation, nor does it prove that the particular mechanism is the 

 correct one. 



In view of the considerable progress made in the isolation in solution of the enzymes 

 involved in the ATP/ADP and the ATP-Pi exchange reactions (see Plaut, Fed. Proc. 

 16, 233, 1957 and Lehninger, Rev. mod. Physics, 31, 144, 1959) it is possible to state, 

 with certainty, that the phosphorylation of ADP and, with reasonable certainty, that 

 the formation of 'high-energy' phosphate do not directly involve the respiratory 

 carriers. In other words, a site of oxidative phosphorylation in the respiratory chain 

 appears to have lost a clear meaning; we may now only attempt to identify the 

 oxidation-reduction couple responsible for conserving the energy which ultimately 

 allows the formation of a 'high-energy' bond. Even this identification is obscured by 

 the conclusion based in the succinate-linked reduction of DPN : that energy conserved 

 at one point in the respiratory chain may be used to drive energy-requiring reactions 

 in another portion of the chain. Thus it may be very difficult to assign a particular 

 phosphate bond to a particular site. A hypothetical example of this may be aff"orded 

 by the possibility that a 'high-energy' intermediate of oxidative phosphorylation 

 generated at a particular site which has insufficient energy to phosphorylate ADP, 

 uses this energy to drive more reduced another component (DPN) so that a partial 

 summation of the energies is obtained in the oxidation of DPNH. Even though this 

 proposition has not been established experimentally, its existence as a possibility 

 makes it much more appropriate to focus our attention upon the experimentally- 

 determinable processes, energy conservation and inhibitory interactions (see also 

 Chance and Hollunger, Nature, Lond. 185, 666, 1960). 



Inhibitory interactions and energy conservation need not occur simultaneously (see 

 below), in fact Chance and Williams (Advanc. Enzymol. 17, 65, 1955) have proposed 

 both simultaneous and non-simultaneous mechanisms. In the absence of spectro- 

 scopically distinct forms of the respiratory carriers that indicate that energy conserva- 

 tion has occurred, the first available physical event discernible is that the inhibition of 

 electron transfer has occurred. 



Added in proof . The idea that the energy of a phosphate bond can be assigned to a 

 particular half of the oxidation-reduction cycle of a carrier is unnecessarily restrictive 

 and the so-called 'carry-over' mechanism proposed by Chance, Williams, Holmes and 

 Higgins (J. Biol. Chem. Ill, 439, 1955) is probably a general mechanism of oxidative 

 and photosynthetic phosphorylation. Designating the energy conserved in oxidation 

 by an asterisk (*) and the total conserved following reduction by a 'squiggle' (~). we 

 write the following equations for DPN- /^-hydroxybuty rate (/>OHB) interaction, the 

 first being the energy-transfer reaction of Equation 7 above. 



DPNH -- 1 -t- X -^ DPNH I -I- X ~ I (18) 



the second an energy-conserving oxidation reaction : 



DPNHI -I- fp -^ DPN*I + rfp (19) 



and the third an energy-conserving reduction: 



DPN*I -f- iiOHE -^ DPNH ~ I + AcAc (20 



The point in the oxidation-reduction cycle at which the bond between the carrier and 

 I is strong enough to inhibit the oxidation-reduction reaction will determine whether the 

 reduced or the oxidized carriers accumulate in state 4. Thus, if the reduced forms 

 accumulate, the ~ form would be involved in the rate-limiting reaction with X. If the 

 oxidized forms accumulate, the * form would be involved in the reaction with X. It is, 

 however, unlikely that both the * and the — forms would be simultaneously involved 

 in the X reaction, since only one form would have a higher energy content than the 

 other, and the reaction with the form of greater energy content would be the preferred 

 one. 



