Reconstitution of oxidative phosphorylation and the adenosine triphosphate-dependent transhydrogenase activity by a combination of membrane fractions from uncA⁻ and uncB⁻ mutant strains of Escherichia coli K12

1. Membrane preparations from both uncA⁻ and uncB⁻ mutant strains of Escherichia coli K12, in which electron transport is uncoupled from phosphorylation, were fractionated by washing with a low-ionic-strength buffer. The fractionation gave a `5mm-Tris wash' and a `membrane residue' from each strain. This technique, applied to membranes from normal cells, separates the Mg²⁺,Ca²⁺-stimulated adenosine triphosphatase activity from the membrane-bound electron-transport chain and the non-energy-linked transhydrogenase activity. 2. Reconstitution of both oxidative phosphorylation and the ATP-dependent transhydrogenase activity was obtained by a combination of the `membrane residue' from strain AN249 (uncA<sup>−</sup>) with the `5mm-Tris wash' from strain AN283 (uncB⁻). 3. Valinomycin plus NH₄⁺ inhibited oxidative phosphorylation both in membranes from a normal strain of E. coli and in the reconstituted membrane system derived from the mutant strains. 4. The electron-transport-dependent transhydrogenase activity was located in the membrane residue and was de-repressed in both the mutant strains. 5. The spatial and functional relationships between the proteins specified by the uncA and uncB genes and the transhydrogenase protein are discussed.