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

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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, 1 mol of ferredoxin was found to react with about 8 mol of p-chloromercuribenzoate in 10 min. Under low ionic strength conditions (1 mM NaCl), the second-order rate constants of this reaction determined spectrophotometrically at 420 and 250 nm were 3,640 and 3,690 M−1 S−1, respectively; under high ionic strength conditions (100 mM NaCl), these rate constants were 1,360 and 1,270 M−1 S−1, respectively. In the presence of the reductase, the rate constants under low and high ionic strength conditions were 54 and 1,040 M−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 3.8 mol 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.