Oxidation of NAD(P)H in a Reconstituted Spinach Chloroplast Preparation Using Ascorbate and Hydrogen Peroxide.

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.