Studies on the Ferredoxin-Ferredoxin-NADP Reductase Complex: Kinetic and Solvent Perturbation Studies on the Location of Sulfhydryl and Aromatic Amino Acid Residues

Abstract

The molecular weight of spinach ferredoxin-NADP reductase [EC 1.6.99.4] was estimated to be 33, 100 by the sedimentation equilibrium method. On the basis of this molecular weight, the amino acid composition of the reductase was determined.
The reactivity of ferredoxin toward p-chloromercuribenzoate was investigated. By measuring the time course of the reaction, 1mol of ferredoxin was found to react with about 8mol of p-chloromercuribenzoate in 10min. Under low ionic strength conditions (1mM NaCl), the second-order rate constants of this reaction determined spectrophotometrically at 420 and 250mn were 3, 640 and 3, 690M^-1•S^-1, respectively; under high ionic strength conditions (100mM NaCl), these rate constants were 1, 360 and 1, 270M^-1•-S^-1, respectively. In the presence of the reductase, the rate constants under low and high ionic strength condi-tions were 54 and 1, 040M^-1•S^-1, respectively.
By investigation of the solvent perturbation effects on the aromatic amino acid residues with 20% ethylene glycol, it was found that ferredoxin, ferredoxin-NADP reductase, and the complex between these proteins had 2.8, 6.3, and 3.8mol of exposed tyrosyl residues per mol of protein, respectively. It therefore seems likely that about 5 tyrosyl residues may exist in the neighborhood of the binding site of the complex of these proteins.