Studies on Nicotinamide Adenine Dinucleotide Phosphate Reductase of Spinach Chloroplasts

The enzyme NADP+ reductase from spinach chloroplasts was studied by various methods of enzymology and protein chemistry. Polyacrylamide gel electrophoresis in sodium dodecyl sulfate showed the polypeptide molecular weight to be 37,000, a value which is almost exactly the same as the minimum molecular weight per FAD, so the enzyme has one FAD per polypeptide chain. Considering the previously reported sedimentation coefficients, the enzyme is probably a monomer. The copper protein plastocyanin, also from spinach was studied by the same method and found to have a polypeptide molecular weight of 10,000, which is the same as the previously reported minimum weight per copper and half the value previously determined by sedimentation, so plastocyanin is a dimer with one copper per polypeptide. The effect of pH and ionic strength on a reaction catalyzed by NADP+ reductase has been studied. The Km for NADPH showed the effects of ionizations at pH 6.3 and 8.9, the former assigned to the substrate and the latter to the enzyme. Vmax was relatively pH-independent from pH 4.5 to 10.5. Km increased linearly with increasing ionic strength. The rates of inactivation of the enzyme by various reagents specific for sulfhydryl groups have been determined. The inactivation was slow compared to other sulfhydryl enzymes, but could be accelerated by NADPH or retarded by NADP+. The inactivation was accompanied by the slower dissociation of the FAD. The inactivation could be reversed by sulfhydryl compounds, but only before the dissociation of FAD occurred. By the use of Ellmann's reagent in the presence of sodium dodecyl sulfate, the enzyme has been found to have a total of eight sulfhydryls, none of which are reactive in the absence of the denaturant. The flavin of NADP+ reductase has been titrated with NADPH, dithionite, and methyl viologen, and photoreduced with ethylenediaminetetraacetic acid as the electron donor. Below pH 9, the NADPH titration produced a semiquinone intermediate form. The NADPH titration above pH 9 and the other methods of titration produced no semiquinone. The reduction potentials for the 1-electron reduction to the semiquinone or the 2-electron reduction directly to the fully reduced form of the flavin were determined. At pH 7, the potentials for the 1-electron steps for the reduction of oxidized to semiquinone and semiquinone to fully reduced forms of the enzyme are -0.32 and -0.40 volt, respectively, values which are intermediate between the potentials of ferredoxin and NADP+.