Oxygen inhibition of photosynthesis was studied with intact spinach (Spinacia oleracca L.) chloroplasts which exhibited very high rates of photosynthetic CO2 reduction and were insensitive to additions of photosynthetic intermediates when CO2 was available at saturating concentrations. Photosynthetic rates were measured polarographically as 02 evolution, and the extent of the reduction of substrate was estimated from the amount of 02 evolved. With CO2 as substrate, inhibition of photosynthesis by 02 was dependent on pH. At pH values above 8, rates of 02 evolution were strongly inhibited by 02 and only a fraction of the added bicarbonate was reduced before 02 evolution ceased. The extent of 02 evolution declined with increasing 02 concentration and decreasing initial bicarbonate concentration. At pH 7.2, the initial photosynthetic rate was inhibited about 30% at high 02 levels, but the extent of 02 evolution was unaffected and most of the added bicarbonate was reduced. Photosynthetic 02 evolution with 3-phosphoglycerate as substrate was similarly dependent on pH and 02 concentration. In contrast, there was little effect of 02 and pH on oxaloacetate-dependent oxygen evolution. Acid-base shift experiments with osmotically shocked chloroplasts showed that ATP formation was not affected by 02. The 23). Oxygenation of RuDP and CO2 evolution from the reaction products would decrease the yield of photosynthesis. Glycolate formation related to the 02 inhibition of photosynthesis has also been explained by oxidative interactions with C6-or Crsugarphosphates (9,16,22).It has been shown that photosynthetic CO2 reduction by isolated chloroplasts is also inhibited by 02 (8,9,21). In the present work, we have studied the effect of 02 on photosynthesis by intact chloroplasts, capable of very high rates of photosynthesis. The aim of our experiments was to gain further insight into the problem of 02 inhibition of photosynthesis in a system less complex than the intact leaf and more sophisticated than a simple enzyme system.
MATERIALS AND METHODSSpinach Hybrid 102 (Spinacia oleracea L.) was purchased from Arthur Yates and Co., Sydney, who obtained it from Alf. Christianson Seed Co., Mount Vernon, Wash. Seeds were germinated in vermiculite and either transferred to hydroponic culture or grown in vermiculite. The nutrient solution was the same as used by Tsui (24), except that sequestrene iron chelate (15.4 mg/l) replaced iron tartrate. The plants were grown for 3 to 4 weeks before the leaves (length 9-10 cm) were harvested. The detached leaves were preilluminated for 5 to 10 min at 4 C in white light (11) and chloroplasts were isolated by a modification (11) of the method of Jensen and Bassham (18).Photosynthetic 02 evolution was measured polarographically at 20 C, using CO2, PGA, or OAA as substrate. Chloroplasts (100-200 ,pg Chl) were suspended in 2.5 ml of a buffer containing 300 mm sorbitol, I mM MgCI2, I mm MnCI2, 2 mM EDTA, 10 mM NaCI, 0.8 mm inorganic phosphate (KH2PO4), and 50 mM HEPES, pH 6.8 to 8.5. CO2 was remo...
