THE MECHANISM OF COENZYME Q REDUCTION IN HEART MITOCHONDRIA 257 



reduction of iron by known redox components of the electron transport 

 chain after the enzyme is denatured by ethanol can be excluded in a 

 number of ways. Since in ETP the same amount of iron is reduced either 

 in the presence of antimycin or anaerobically, it follows that cytochromes 

 c^, c and a cannot participate in this reduction. The succinic-CoQ reduct- 

 ase does not contain CoQ, and the cytochrome b present is not reduced; 

 therefore, neither of these components reduces the non-haem iron during 

 its extraction from the denatured protein. The possible interference of the 



TABLE III 



Reduction of Xon-Haem Iron in Mitochondrial .Subfractions* 



™ , Percentage of total non- 



„ , haem Fe reduced 



rreparation non-haem 



Fe 



by DPXH by succinate 



Primary succinic flavoprotein [4] 17 o o 



Succinic-CoQ reductase [5] 34 o 23 



Succinic-cytochrome c reductase [9] 22 < i 23 



DPNH-cytochrome f reductase [10] 15 30 <2 



ETP„ [11] 9 39 30 



* The procedure for measuring the redox state of enzyme-bound non-haem 

 iron will be described in detail elsewhere [12]. A brief summary of the method is 

 as follows : (i) The preparation is treated with KCN or antimycin A to block 

 oxidation ; (ii) substrate is added at zero time ; (iii) the reaction is stopped by adding 

 CdCh ; (iv) the reduced non-haem iron is extracted with a mixture of ethanol (70*^0), 

 o-chloromercuriphenol (5 mg./ml.), sodium acetate (100 /tmolesml., pH 4 "6), 

 and bathophenanthroline (o-i mg./ml.); (v) the ferro-bathophenanthroline colour 

 is measured at 535 m/i against a control to which substrate was added after the 

 CdClo. 



The cadmium ions [13] and the organic mercurical are necessary to prevent 

 the non-enzymic reduction of iron by thiols. 



fiavoproteins cannot be entirely eliminated, but in most preparations the 

 amount of iron reduced is considerably in excess of the fiavoproteins and 

 the non-haem iron is reduced in the succinic-CoQ reductase but not in 

 the primary succinic flavoprotein. The ratio of flavin to protein in the 

 latter two preparations is identical, which also indicates that non-enzymic 

 reduction of the iron by reduced flavoprotein does not occur. 



Amytal, a specific inhibitor of DPXH oxidase, blocks the reduction of 

 non-haem iron by DPXH (Table IV), but malonate increases the total 

 amount of iron reduced by this substrate. The reverse is true with suc- 

 cinate as substrate. Malonate blocks reduction of iron by succinate in 

 ETP, but amytal increases the amount of iron reduced by succinate. In 

 the presence of both succinate and DPXH the total amount of iron 



VOL. II. S 



