262 DANIEL M. ZIEGLER 



respiratory chain preparations but not in the extracted, soluble form ; second, that 

 it has two reaction sites for phenazine methosulphate in particulate preparations 

 but only one in soluble ones and thus in the particulate form the enzyme has twice 

 the Qo, in the phenazine assay that it has in soluble preparations; and, third, that 

 in particulate preparations, but not in purified, soluble ones, it reacts with methy- 

 lene blue, brilliant cresyl blue, and related dyes. In regard to all three criteria the 

 CoQ-reductase of Ziegler and colleagues behaves like a respiratory chain prepara- 

 tion, as expected from the fact that the enzyme is still linked to cytochrome b in 

 this particle. Work in our laboratory suggests that those properties of succinic 

 dehydrogenase which are lost on solubilization are not fundamental characteristics 

 of the flavoprotein itself but may be the consequence of the binding of some of 

 the non-haem iron of the flavoprotein to the respiratory chain. We proposed some 

 years ago that at least two of the four iron atoms of the isolated enzyme may act as 

 ligands of the flavoprotein to the respiratory chain in particulate preparations. It 

 is established that at least two of the four irons in the isolated flavoprotein do not 

 function in oxido-reduction in purified preparations, although they might do so 

 when bound in a particle. If so, they might also be involved in the catalytic cycling 

 of CoQ in such particulate preparations as the CoQ reductase. 



The anomalous absorption changes in the flavin region which Dr. Ziegler has 

 observed, are, probably not fortuitously, very similar to those which occur in 

 a-glycerophosphate dehydrogenase, a flavoprotein rich in iron, in the succinic 

 dehydrogenase of Micrococcus lactilyticiis, where iron has been shown to undergo 

 oxido-reduction by the substrate, and in a rat liver enzyme which oxidizes inositol 

 and which doesn't even have flavin but is an iron enzyme. These considerations 

 would again suggest that part of the iron complement of the flavoprotein might 

 undergo oxido-reduction in respiratory chain preparations. 



Ziegler: The properties of the soluble succinic CoQ-reductase appear to be 

 identical with those of the particle-bound dehydrogenase; the phenazine metho- 

 sulphate reductase activity of the isolated enzyme is partly sensitive to cyanide. 

 Cyanide also blocks the reduction of Q. We have tested a number of compounds 

 that have been used to inhibit the particle-bound dehydrogenase, and disulphide 

 compounds such as lipoic acid are very effective inhibitors of Q reduction. 



The discrepancy between the concentration of iron in the enzyme reported 

 here and in our earlier publication is due to a change in the method of estimating 

 enzyme-bound iron. The ratio of 4 irons per flavin was obtained on a preparation 

 that had been thoroughly dialyzed against a versene solution. However, the activity 

 of the enzyme is destroyed by prolonged dialysis. Currently we remove extraneous 

 iron by passing the enzyme through a column of Dowex A-i chelating resin. This 

 procedure does not destroy the Q-reductase activity of the enzyme and the ratio 

 of non-haem iron to flavin is consistently 8:1. 



Ernster : I would like to hear how you visualize the relationship of this 

 mechanism to that prevailing in phosphorylating preparations, especially with 

 respect to the participation of cytochrome b. 



Ziegler: I have discussed this previously with Dr. Ernster and I think we are 

 in full agreement. Could he put the mechanism we discussed on the board ? 



Ernster : Well, all I meant to ask is this : is this form of succinic dehydrogenase, 

 which is now a Q-reductase, a cytochrome b reductase as well ? 



