40] Ferredoxin-NADP reductase from spinach

Abstract

Publisher Summary This chapter discusses the determination of ferredoxin–NADP reductase from spinach. Ferredoxin–NADP reductase is a chloroplast flavoprotein and catalyzes the reduction of NADP with reduced ferredoxin. The crystalline enzyme shows not only ferredoxin–NADP reductase activity, but also a number of other enzymatic activities, such as NADPH diaphorase, transhydrogenase, and hemoprotein reductase activity. Ferredoxin–NADP reductase catalyzes reversible electron transport between ferredoxin and NADP. The usual assay method is based on the reduction of NADP by reduced ferredoxin in the presence of a regenerating system, either illuminated chloroplasts or the H 2 –hydrogenase system in the dark. The assay method for the reverse reaction involves the reduction of cytochrome c , as the terminal electron acceptor, in the presence of ferredoxin and NADPH. During purification of the enzyme, the NADPH-diaphorase activity serves as a routine assay. The ferredoxin–NADP reductase has a molecular weight of about 40,000, contains one flavin adenine dinucleotide (FAD) per mole, and shows a typical flavoprotein absorption spectrum with absorption maxima at 275, 385, and 456 nm and minima at 321 and 410 nm.