Mechanism of the Photochemical Activity of Isolated Chloroplasts: VII. Inactivation of a Photoact by Dilute Ferricyanide Ion.

Destructive effects of the most commonly used Hill oxidant, ferricyanide ion, are well documented and include its acceleration of the second order inactivation process in isolated chloroplasts (3). Concentrated ferricyanide (50 mM), especially at pH values above 7, rapidly uncoupled photophosphorylation in spinach chloroplasts (4), thus accounting for its increase in the light-independent rate constant of ferricyanide reduction kD by chard and beet chloroplasts (1, 9), and it strongly inactivated a light step associated with oxygen evolution (4), which would account for our lowered photochemical efficiency as kL (1, 9). Ferrocyanide ion was equally effective (4), and the effect on kL was obtained with all artificial oxidants tried (9). The less concentrated (0.5 mM) ferricyanide ordinarily used to measure the velocity of the Hill reaction also inhibited photophosphorylation below pH 5.5 by potentiating acid lability and by solubilizing the coupling factor CF1 as did ferrocyanide (12). In addition it partially inactivated, independent of pH, a dark step associated with the oxygen-evolving site, but this was prevented by weak illumination and by ferrocyanide, as it was an oxidative effect of ferricyanide (4). We now report, by way of kinetically resolved kL (9, 13) with chloroplasts from a cultivar of sugar beet under given conditions of measurement, that the limiting photochemical step can also be very labile toward the lower concentration of ferricyanide, and that this characteristic is promoted, as uncoupling was (4), by acidic as well as basic solutions. Chloroplasts were prepared from leaves of field-grown sugar beets by brief blending and centrifugal washing in 0.5 M sUcrose, 10 mM NaCl and 50 mm Tricine buffer at pH 7.8, and by final suspension in the same without buffer for snap-freezing and storage at -55 C. The routine 1-ml reaction mixture contained 100 ,tmoles of sodium phosphate buffer of various values of pH, 10 ,umoles NaCl, 0.375 ,umole of potassium ferricyanide with 0.125 ,umole potassium ferrocyanide to poise the initial potential, or with 10-fold concentration of this couple with or without added DPIP4, and chloroplast suspension containing 0.10 mg of chlorophyll with 50 ,tmoles of sucrose. Ferricyanide reduction was recorded potentiometric-