Enzymology of Ubiquinone‐Utilizing Electron Transfer Complexes in Nonionic Detergent

The enzymology of isolated succinate: ubiquinone reductase and ubiquinone: cytochrome c reductase in nonionic detergents (alkyl polyoxyethylene derivatives) was studied. In the membrane the two multiprotein complexes and their hydrophobic substrates ubiquinone and dihydroubiquinone, are embedded in a common lipid bilayer. In detergent solutions the complexes are each inserted into micelles. Detergent micelles also serve as a solvent for the complexes hydrophobic substrates. As a consequence the isolated complexes are in a discontinuous phase with respect to their hydrophobic substrates and with respect to each other. Three types of assays were used. Firstly, single enzyme assays in which the hydrophobic substrates had to transfer from free micelles to the complex‐bound micelles in order for enzymic reactions to occur. Secondly, assays in which the enzymic reactions were coupled to auxiliary non‐enzymic reactions which rapidly converted the hydrophobic products back into substrates within the complex‐bound micelle. Dichloroindophenol was used for the oxidation of dihydroubiquinone and dihydroduroquinone for the reduction of ubiquinone. Thirdly, assays in which the succinate: ubiquinone reductase reaction was coupled with the ubiquinone: cytochrome c reductase reaction. With the first type of assay, the kinetics of the substrate transfer reaction was dependent upon the type of detergent. In detergents with small polyoxyethylene head groups the transfer reactions were rate‐limiting, and in detergents with large polyoxyethylene head groups the transfer reactions were fast and the enzymic reactions were rate‐limiting. With the second type of assay, the substrate concentration within the complex‐bound micelle was controlled by the equilibrium position of the substrate transfer reaction. Half‐maximal activity was obtained when the hydrophobic substrate concentration was half the total micelle concentration. This indicated that the equilibrium constant of the substrate transfer reaction was equal to one. With the third type of assay, detergent micelles containing the hydrophobic product/substrate couple functioned as substrate carriers between the two complexes. The reaction rates depended on the kinetics of the substrate transfer reaction. Direct complex‐complex interaction did not occur.