Spinach chloroplasts, pre-incubated with ferricyanide, acquire the ability to make ATP in the dark provided they are supplied with a reductant and a lipophilic mediator that can penetrate the membrane. The mediator must be of the type that, upon oxidation, releases protons into the surrounding medium such as 2,3,5,6-tetramethyl-p-phenylenediamine (DAD). 2. Dark phosphorylation is not affected by the electron transport inhibitor, 3(3,4-dichlorophenyl)-1,1-dimethyl urea (DCMU) or 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB), but is inhibited by uncouplers of photophosphorylation (e.g. NH4Cl and carbonylcyanide-m-chlorophenylhydrazone (CCCP)) and high concentrations of the energy transfer inhibitor, Dio-9. 3. Because only catalytic amounts of the mediator DAD are required to saturate dark phosphorylation, it is concluded that DAD shuttles reducing equivalents across the membrane from the reductant, ascorbate, on the outside to ferricyanide, the oxidant, trapped on the inside. 4. The results are interpreted within the framework of the chemiosmotic hypothesis for the coupling of electron transport to phosphorylation.
