Hydrogen Peroxide Synthesis in Isolated Spinach Chloroplast Lamellae

Abstract
Light-dependent O2 reduction concomitant with O2 evolution, ATP formation, and NADP reduction were determined in isolated spinach (Spinacia oleracea L. var. America) chloroplast lamellae fortified with NADP and ferredoxin. These reactions were investigated in the presence or absence of catalase, providing a tool to estimate the reduction of O2 to H2O2 (Mehler reaction) concomitant with NADP reduction. In the presence of 250 micromolar O2, O2 photoreduction, simultaneous with NADP photoreduction, was dependent upon light intensity, ferredoxin, Mn2+, NADP, and the extent of coupling of phosphorylation to electron flow. In the presence of an uncoupling concentration of NH4+, saturating light intensity (>500 watts/square meter), saturating ferredoxin (10 micromolarity) rate-limiting to saturating NADP (0.2-0.9 millimolarity), and Mn2+ (50-1000 micromolarity), the maxium rates of O2 reduction were 13-25 micromoles/milligram chlorophyll per hour, while concomitant rates of O2 evolution and NADP reduction were 69 to 96 and 134 to 192 micromoles/milligram chlorophyll per hour, respectively. Catalase did not affect the rate of NADPH or ATP formation but decreased the NADPH:O2 ratios from 2.3-2.8 to 1.9-2.1 in the presence of rate-limiting as well as saturating concentrations of NADP. Photosynthetic electron flow at a rate of 31 micromoles O2 evolved/milligram chlorophyll per hour was coupled to the synthesis of 91 micromoles ATP/milligram chlorophyll per hour, while the concomitant rate of O2 reduction was 0.6 micromoles/milligram chlorophyll per hour and was calculated to be associated with an apparent ATP formation of only 2 micromoles/milligram chlorophyll per hour. Thus, electron flow from H2O to O2 did not result in ATP formation significantly above that produced during NADP reduction.