596 Discussion 



heart-muscle preparations, under the conditions of concentration and temperature 

 which have usually been employed in studying the succinate oxidase and DPNH 

 oxidase in these preparations. Our recent studies have supported our original view 

 that cytochrome b is involved in succinate oxidation, and not (or to only a small 

 degree) in DPNH oxidation. 



We agree that experiments with low concentrations of DPNH, and with succinate 

 in the presence of cyanide, have shown that in these preparations reducing equivalents 

 can be transferred to the iron atom of cytochrome c without having passed through 

 the iron atom of cytochrome b. We agree with Chance that this is important, especially 

 when taken in conjunction with his discovery that phosphorylating preparations 

 behave differently. We have attempted to account for all these findings in the reaction 

 mechanisms which we propose. 



We believe that the difference in behaviour of cytochrome b in non-phosphorylating 

 preparations to DPNH and succinate (in the absence of cyanide) is significant, and 

 is connected with the fact that, in the phosphorylating system, reduction of cytochrome 

 b by DPNH is associated with phosphorylation, whereas reduction by succinate is not 

 associated with phosphorylation. 



The Influence of Cyanide on the Reactivity of Cytochrome b 

 Estabrook: Slater has introduced the possible inhibitory effect of cyanide in the inter- 

 pretation of Chance's kinetic studies of cytochrome b reduction in heart-muscle 

 preparations. Studies (Estabrook,/. biol. Chem. 227, 1093, 1957; 230, 735, 1958) with 

 either a cytochrome c deficient preparation of liver mitochondria or with a cholate 

 preparation from heart muscle, situations in which electron transport is interrupted 

 because of an absence of cytochrome c or cytochrome oxidase, show the same type of 

 kinetics of cytochrome b, relative to flavoprotein and cytochrome Cj, as reported by 

 Chance. In these studies no cyanide was added to introduce the complication men- 

 tioned by Slater. These kinetics when analysed using an analogue computer simul- 

 taneously solving the differential equations represented for a 'straight-chain' or a 

 'split-chain' mechanism are compatible only with the 'split-chain' mechanism inferring 

 a by-pass of cytochrome b. 

 Slater: Estabrook's conclusion that cytochrome b is by-passed in the reduction of cyto- 

 chrome c by DPNH is, of course, in complete agreement with my earlier observations, 

 and with our newer observations with low concentrations of DPNH. These were also 

 made in the absence of cyanide. His comment is therefore irrelevant to the discussion 

 with Chance, which concerned experiments with succinate as substrate. 



We were naturally aware of Estabrook's important observations, which can in fact 

 be explained more satisfactorily by the mechanism which we propose than by simpler 

 'straight-chain' or 'split-chain' mechanisms. Estabrook has shown that not only is 

 cytochrome b by-passed in the reaction between DPNH and cytochrome c, but also 

 cytochrome c^, since in the absence of added cytochrome c only flavoprotein is reduced 

 at a rate sufficient to explain the reduction of added cytochrome c. This can be under- 

 stood if the fpH radical produced in reaction (13) reacts more rapidly with added 

 cytochrome c than with the endogenous cytochrome Cj, which seems likely. 



