Reconstitution of Respiratory Chain Enzyme Systems

The current concept of the respiratory chain enzyme system enunciates the orderly transfer of hydrogen (electron) from one respiratory carrier to the next with molecular oxygen as the final electron acceptor.Thus, in the succinate oxidase system, the transfer can be depicted essentially as Succinate 4 succinate dehydrogenase + cytochrome b + Cl + c + a + u3 + 02 (Here, b, cl, c, a, and a3 are cytochromes and the arrows indicate the flow of electrons.)It is true that workers in the field are not in agreement on the components and the sequence of the electron transfer in the oxidation of succinate or DPNH by the respiratory chain.These investigators differ especially with respect to the converging point of DPNH oxidase and succinate oxidase; the function of ubiquinone (coenzyme Q), nonheme iron, and copper; and the identity of cytochromes a and a+.On the other hand, all the experimental results documented since 1925 support the concept and indeed strengthen it to such a position that functional behaviors may be almost equated with structural constitution.In this laboratory, a project has been undert.aken in the sequential fragmentation of the respiratory chain and systematic reconstitution from the components obtained.It is hoped that postulates of biological organization can then be more fully explored on the physicochemical basis and in turn the functional as well as structural manifestations of intracellular organelles may be more rationally pursued.We have fragmented the succinate oxidase system into two parts: one from succinate to cytochrome c, the other from cytochrome c to molecular oxygen.The former has been further disintegrated into succinate dehydrogenase and the cytochrome b-cl particle.This communication deals with the methods for the preparation of succinate-cytochrome c reductase (i.e. the portion of the chain that transfers electrons from succinate to cytochrome c) and its fragmentation into the cytochrome b-cl particle and soluble succinate dehydrogenase.Some properties of these segments are also described.The subsequent paper (2) reports the reconstitution of succinate-cytochrome c reductase from soluble succinate dehydrogenase and the cytochrome b-cl particle.