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A spinach (Spinacia oleracia var. America) chloroplast particle fortified with ferredoxin, fructose-1,6-bisphosphate, or ribose-5-phosphate and NADP has been shown to generate NADPH by the oxidation of glyceraldehyde-3 phosphate to glycerate-3-phosphate (PGA) and to reduce ferredoxin with the NADPH. The resulting reduced ferredoxin can reduce O(2) to H(2)O(2), nitrite to ammonia, or protons to H(2). Hydrogen production was the result of adding hydrogenase from Chlamydomonas reinhardii to the chloroplast preparation. The predicted stoichiometry of 1 PGA:1 O(2) in the absence of and 2 PGA:1 O(2) in the presence of catalase was observed indicating H(2)O(2) as the end product of O(2) reduction. The predicted stoichiometry of 3 PGA:1 nitrite:1 ammonia was also observed. A scheme is presented to account for a sustained generation of NADP and ATP necessary for the dissimilation of starch in the darkened chloroplast. The unifying term chloroplast respiration is introduced to account for those reactions in which reduced ferredoxin interacts with physiological acceptors other than NADP or nitrite, hydrogen, or O(2) respiration when nitrite, protons, or O(2) is the ultimate electron acceptor.

The conversion of fructose-1,6-bisphosphate to glycerate-3-phosphate (PGA) was studied in a reconstituted spinach (Spinacia oleracea L.) chloroplast preparation to determine whether a chloroplast-localized NAB(P)H-oxidizing system (Kow, Smyth, Gibbs 1982 Plant Physiol 69: 72-76 with substrates of ascorbate, NAD(P)H, and H(2)O(2) could serve as a coupling enzyme in the recycling of NAD(P)H. The rate of PGA formation was monitored as an indicator of NAD(P) generation. With NAD as a cofactor, ascorbate enhanced PGA formation, and an additional increase resulted upon addition of glucose-glucose oxidase, a H(2)O(2)-generating enzyme. This increase in PGA formation due to H(2)O(2) was eliminated by the addition of catalase. With NADP and ferredoxin as cofactors, the recycling of NADP apparently was catalyzed both by ferredoxin-NADP reductase coupled to O(2) and by the NAD(P)H-oxidizing system.It was concluded that the oxidation of NAD(P)H by a system using ascorbate and H(2)O(2) can serve as a means of recycling NAD(P)H but that another reaction involving ascorbate and NAD(P)H may also function in the spinach chloroplast.

A particulate preparation (MgP) capable of photosynthetic CO(2) assimilation without the addition of stromal protein was obtained by rupturing whole spinach (Spinacia oleracea var. America) chloroplasts in 15 millimolar MgCl(2) buffered with Tricine at pH 8.5. This CO(2) assimilation was dependent upon light, inorganic phosphate, ferredoxin, ADP, NAD or NADP, and primer. Excepting glycolate, the products of CO(2) fixation by MgP were similar to those found with whole chloroplasts.Glycerate-3-phosphate (PGA), fructose-1, 6-bisphosphate (FBP), and ribose-5-phosphate (R5P) but not fructose-6-P (F6P) functioned as primers. Concentrations of PGA and FBP but not of R5P higher than 2 millimolar were inhibitory to CO(2) fixation. A lag of CO(2) fixation was observed with PGA and FBP but not with R5P. This lag as well as inhibition by NADP, ADP, and ATP in the FBP-primed preparation was eliminated by an equimolar mixture of FBP plus F6P indicating FBPase as the sensitive site. NADP, ADP, and ATP also blocked CO(2) fixation by the PGA-fortified preparation but inhibition was even more sensitive than that observed when FBP was added. Inhibition by AMP in the PGA and FBP-primed preparations was not affected by the addition of F6P. When R5P was the starting primer, inhibition of CO(2) fixation was relatively insensitive to the adenylates and NADP. In contrast to the parent whole chloroplast, CO(2) fixation by MgP was insensitive to high (5 millimolar) inorganic phosphate. Depending upon the ferredoxin concentration, NAD was as effective as NADP in supporting CO(2) fixation.

A NAD(P)H oxidizing system (NAAP) was detected and partially purified from leaves of spinach and Sedum praealtum, seeds and leaves of pea and cells of green and red algae which oxidized NAD(P)H in the presence of ascorbate and H(2)O(2).The partially-purified spinach system had substrate K(m) values of 5 micromolar for NADH, 50 micromolar for H(2)O(2), and 300 micromolar for l-ascorbic acid at the pH optimum of 6.8. NADH was a better electron donor than NADPH. Among other electron donors, isoascorbic acid had considerable activity but hydroquinone and resorcinol had only weak activities. The enzyme was inhibited by cyanide, alpha,alpha'-dipyridyl, and mono-and di-thiol reagents. Inhibition by thiol-reagents was partially restored by Fe(2+) as was enzymic activity lost following dialysis against buffer.Subcellular localization studies with spinach and S. praealtum leaves indicated that a portion of the cell's NAAP was in the chloroplast fraction. Photosynthetic conditions resulted in a decrease in this activity solubilized from spinach and S. praealtum chloroplasts. The presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea or Fe(2+) in the incubation medium eliminated the light-mediated inhibition of NAAP.NAAP may function in the recycling of NAD(P)H generated in the dark within the chloroplast. Inasmuch as all preparations of NAAP contained ascorbate peroxidase activity, the data do not rule out the possibility that NAAP is the same protein as ascorbate peroxidase or, alternatively, a combination of ascorbate peroxidase and some other enzyme.

The light-dependent synthesis of glycolate derived from fructose 1,6-diphosphate, ribose 5-phosphate, or glycerate 3-phosphate was studied in the intact spinach (Spinacia oleracea) chloroplasts in the absence of CO(2). Glycolate yield increased with an elevation of O(2), pH, and the concentration of the phosphorylated compound supplied. No pH optimum was observed as the pH was increased from 7.4 to 8.5. The average maximal rate of glycolate synthesis was 50 mumoles per milligram chlorophyll per hour while the highest rate observed was 92 with 2.5 mm fructose 1,6-diphosphate in 100% O(2). The highest yields of glycolate synthesized from fructose 1,6-diphosphate, ribose 5-phosphate, or glycerate 3-phosphate were 0.14, 0.24, and 0.30, respectively, on a molar basis.