An ATP-driven rise in fluorescence yield (Q reduction) and cytochrome f oxidation in light-triggered chloroplasts are demonstrated. Both effects are shown to be dependent on the creation and maintenance of the light-triggered state. ATP-induced reduction of Q depends upon the presence of an electron donor in the dark with a redox potential up to, but not exceeding, 300 mV. The effects are abolished by uncouplers or energy transfer inhibitors. 3-(3,4-Dichloropheny1)-1,l-dimethylurea inhibits the ATP-driven reduction of Q but not that of cytochrome oxidation. A location for a coupling site and the site of action of electron-transport inhibitors is suggested on the basis of these observations. Energy-dependent reverse electron transport has been clearly demonstrated in mitochondria and provided an extremely useful tool for studying the interaction between the electron transport and energy conserving chains in that system [1,2].I n chloroplasts, all attempts to show energydependent reverse electron flow have not been successful. We have recently reported briefly [3] that ATP-driven reverse electron transport can be seen in chloroplasts by following the change in the fluorescence yield of chlorophyll, in a system lighttriggered to catalyse ATP hydrolysis.This communication describes this system in further detail, demonstrating the effect of a variety of treatments, and reports moreover the simultaneously observed energy-dependent oxidation of cytochrome f in the chloroplast.
MATERIALS AND METHODSChloroplasts from lettuce (Latuca satiwa var. romaine) were prepared as previously described [4], except for final resuspension, which was in 0.2M sucrose, 0.1 M KCl. The chloroplasts were stored in concentrated suspension in the sucrose-KC1 medium. For fluorescence measurements, light-triggering was carried out in 1-cm-square cuvettes with 1.8 ml of a standard reaction mixture containing 90 mM sucrose; 45 mM NaC1; 22 mM Tris-HC1, pH 7.8; dithiothreitol, 5 mM; 5 mM MgC1,; phenazine methansulfate, 2.5 pM and chloroplasts containing around 40 pg chlorophyll/ml. The mixture was light-triggered for Definitions. Q, the fluorescence quenching primary electron acceptor of photosystem 11; dA,,,-,,,, the change of absorbance of a suspension at 554 nm minus the change at 540 nm, when measured in a l-cm pathlength cell. ~Q A T P , percentage change in reduction of Q induced by ATP.2 min by illumination with a xenon lamp filtered through 2-cm saturated CuSO, solution resulting in an intensity of about 6 x 106 ergs x cm-a x s-l a t the cuvette surface. I n experiments where a dilution procedure was employed, the light-triggering was carried out with the same quantity of chloroplasts contained in 0.18 ml of reaction mixture and 1-2 s before the end of the light-triggering period the volume was brought up to 1.8 ml with reaction mixture (lacking chloroplasts).Within a few seconds after turning off the triggering light, the fluorescence-exciting light was admitted, and measurements commenced. Fluorescence measurements were made in an apparat...
