608 B. Chance 



believed to be at hand to indicate their participation in the respiratory chain 

 of phosphorylating mitochondria of heart, Uver and kidney tissues are : 



succinate -^ fp 

 \ 



/j ^ Ci — ^ c — ^ a — ^ flg -^ O2 (6) 



/ 

 DPNH--fp 



t 

 Amytal antimycin-A CN, CO 



For the intact chain, no antimycin-A sensitive factor other than cytochrome 

 b need be postulated, and no postulate of an Amytal-sensitive factor between 

 DPNH and flavoprotein has yet been made (Chance, 1956). 



Since direct data do not yet show the site of action of the quinone in 

 relation to cytochrome h in the phosphorylating preparations, we do not 

 include it at present. Hatefi, Lester, Crane and Widmer (1959) maintain 

 that coenzyme Q is located on the oxygen side of cytochrome h, presumably 

 between cytochromes b and q or c. This result is difficult to reconcile with 

 previous results, with those on other preparations (Green, 1959), and with 

 the titration data on non-phosphorylating preparations (see above). 



Possible Ligands Associated with Phosphorylation 



On the basis of steady-state and kinetic tests, electron transfer in phosphory- 

 lating preparations is specifically blocked in the absence of ADP or inorganic 

 phosphate (Lardy and Wellman, 1952; Chance, 1956). Since neither ADP nor 

 inorganic phosphate is required for electron transfer in the Keilin and Hartree 

 preparation (Bonner, 1954) it is concluded that a substance, I, inhibits electron 

 transfer {Note 2). It was further suggested by Chance and Williams (1956b) 

 that the substance I may not be identical for the sites of energy conservation; 

 and Slater (1958) identifies I^, Ig, I3, which we can label I^, I^ and 1,^ to indicate 

 the respiratory component with which the interaction occurs (Chance, 1959a). 

 An intermediate of phosphorylation, X, formed upon addition of ADP and 

 inorganic phosphate (Pi), reverses the inhibition caused by I by combining 

 with it and transferring conserved energy ultimately to ADP and Pi. Electron 

 transfer can then proceed at a rate set by the regeneration of I or X or by the 

 rate of the intercarrier reactions {Note 3). 



Evidence for such intermediates can be readily indicated by a comparison 

 of the rates at which ADP and an uncoupling agent (dicoumarol) can change 

 the steady-state oxidation-reduction level of cytochrome c in a preparation 

 of phosphorylating mitochondria (Fig. 5). The aerobic suspension of 

 mitochondria at 10°C is treated with substrate (succinate) and 250 //m ADP 

 causes a readily measurable change of the steady state in the direction of 

 increasing reduction at a rate of 01 /^moles of Fe 1.^ sec^^. After ~100 sec, 



