66] Delineation of the mechanism of ATP synthesis in chloroplasts: Use of uncouplers, energy transfer inhibitors, and modifiers of coupling factor 1

Abstract

This chapter discusses the role of uncouplers, energy transfer inhibitors, and modifiers of coupling factor 1 in ATP synthesis in chloroplasts. A large number of reagents and treatments affect photophosphorylation. Inhibitors of electron flow abolish ATP synthesis because they also block the formation of the electrochemical proton gradient, the driving force for ATP synthesis. ATP synthesis may also be uncoupled from electron flow. Reagents that enhance the permeability of thylakoid membranes to protons uncouple phosphorylation from electron flow are called uncouplers. Lipid-soluble weak acids, such as carbonyl cyanide m -chlorophenylhydrazone (CCP), which act as proton carriers across both natural and synthetic membranes, are uncouplers. Some uncouplers are destroyed by illumination in the presence of thylakoids. Gramicidin is not a proton carrier, but instead, forms an aqueous transmembrane channel through which protons and other small ions can pass. N , N '-Dicyclohexylcarbodiimidc (DCCD) and phlorizin 4,4′,6′-trihydroxy-2′-glucosidodihydrochalconc) inhibit photophosphorylation, but interact with different parts of the ATPase complex. Phlorizin is the only inhibitor of this type which is available commercially. Because phlorizin acts rapidly, it may be added directly to reaction mixtures. Phlorizin specifically inhibits electron flow which is dependent on phosphorylation.