The role of copper in photosynthetic organisms depends greatly on its concentration. Copper, as cupric ion, in trace amounts is an essential micronutrient for algae and higher plants (17,20) and is an essential constituent of several enzymes such as polyphenol oxidase (3) and plastocyanin (8), a component of photosynthetic electron transport. Concentrations higher than 1 /tm are increasingly toxic to algal and higher plant tissues (4, 13). Cupric sulfate has been extensively used as an algaecide since the beginning of the century (12). The cupric ion has been shown to be an inhibitor of photosynthesis in algal cells (4,11,16) and to inhibit photosynthetic electron transport in isolated chloroplasts (5, 10).The mechanism by which the Cu'+ inhibits the photosynthetic apparatus has been only partially elucidated thus far. The data on inhibition of photosynthesis in isolated chloroplasts gives an incomplete picture of the specific sites of inhibition in the electron transport chain. Macdowall (10) He studied the effect of light intensity on inhibition and concluded that the light reactions were directly affected by Cu21. In addition, Haberman (5) using chloroplasts from Phytolacca americanca showed that both the Hill and Mehler reactions were inhibited by Cu2+ but that the Mehler reaction was inhibited by lower concentrations of the ion.In contrast to Macdowall, Haberman concluded that Cu2+ was inhibiting a dark reaction and did not alter the reactions associated with the photoacts. She observed that Mn2+ added at a concentration of 0.5 mm reduced the inhibition of chloroplasts by Cu2+ and postulated that the Cu2+ was affecting the site of manganese function in 02 evolution. Recently exogenous Mn2+ has been shown to donate electrons after the water oxidation site of photosystem II of isolated chloroplasts (2), suggesting that, at high concentrations, manganese functions in a way other than its primary role in the water oxidation act.This work was conducted with the purpose of resolving the sites of Cu2+ inhibition in photosynthetic electron transport. Since previous work and our preliminary experiments pointed to the fact that photosystem II is preferentially inhibited by Cu2+ emphasis was given to finding the specific sites of inhibition within this photosystem.
MATERIALS AND METHODSChloroplasts were prepared from market-grown spinach (Spinacia olercacea L.) as described by Robinson and Stocking (14). MnCl2 was excluded from the grinding and resuspending media in the preparations where Mn2+ was used as an electron donor. Chlorophyll was determined by the method of Arnon (1). Ferredoxin was isolated by the method of San Pietro (15) as modified by Swader and Jacobson (18). The 02 evolving capacity of the chloroplasts was destroyed by a mild heat treatment as described by Hinkson and Vernon (6). The chloroplasts were uncoupled by adding ammonium ions following the principle discovered by Krogman et al. (9).The normal reaction media for studying 02 evolution had the following composition in a total volume of 2...