Light dependent oxygen metabolism of chloroplast preparations. III. Photooxidation of ascorbic acid

Abstract

Ascorbic acid is a normal constituent not only of the chloroplasts but also of the other protoplasmic components of the cells of green leaves. In spite of its known widespread occurrence in plants, few functions of ascorbic acid in plant metabolism have been established (see review by Mapson, 15). The participation of ascorbic acid as an intermediate hydrogen carrier in photosynthesis has been suggested (16, 17) and certainly the experiments of Krasnovsky on the photochemical reduction of chlorophyll in vitro by ascorbic acid (13, 14) have added new interest to these suggestions.. Ascorbic acid was at one time considered to be ,a necessary component of the photosyntheticf phosphorylation system but more recently it has been regarded as having a protective role in preventing inactivation of essential components of the chloroplasts (Arnon, 1). Soon after he discovered that chloroplasts could use oxygen as a Hill reagent, Mehler (18, 19) reported that rates of net oxygen uptake in the light by reaction mixtures containing chloroplasts, ethanol, and catalase were stimulated several fold if the chloroplasts had first reduced quinone. To determine if it was necessary for chloroplasts to reduce quinone in the light, Mehler added ascorbic acid to the reaction mixture to reduce quinone in the dark. When an excess of ascorbic acid was addeJ, an extremely rapid oxygen consumption occurred on illumnination. The nature of this rapid uptake in the presence of ascorbic acid was of particular interest because of the apparent increase in the efficiency of utilization of light energy. Later, Vernon and Kamen compared the photooxidations of various redox couples by spinach leaf homogenates and sonic extracts of Rhodospirillum rubrum (21). The most active couple which they found was the combination of 2,6-dichlorophenolindophenol (DPIP, acting as an electron carrier) and ascorbic acid (acting as the ultimate electron donor). In both plant and bacterial extracts, oxygen uptake in the light continued until oxygen equivalent to the excess ascorbic acid in the system had been consumed. The nature of this rapid DPIP-mediated photooxidation of ascorbic acid has since been investigated using tracer oxygen techniques (Habermann & Vernon, 10). The results of these studies support the hypothesis that ascorbic acid can be oxidized by the oxidized product of photolysis. This paper describes investigations of the mechanism of the photooxidation of ascorbic acid mediated by chloroplast reaction systems.4 Addition of ascorbic acid during either a Mehler or exchange reaction results in an immediate acceleration of the rate of net oxygen uptake. Just as the rates of the Mehler and exchange reactions are accelerated after a chloroplast preparation has reduced quinone (19, 8), previous reduction of quinone has a stimulating effect on rates Hill reaction (production only):