Binding of ferredoxin to ferredoxin:NADP+ oxidoreductase: the role of carboxyl groups, electrostatic surface potential, and molecular dipole moment.

The small, soluble, (2Fe-2S)-containing protein ferredoxin (Fd) mediates electron transfer from the chloroplast photosystem I to ferredoxin: NADP+ oxidoreductase (FNR), a flavoenzyme located on the stromal side of the thylakoid membrane. Ferredoxin and FNR form a 1:1 complex, which is stabilized by electrostatic interactions between acidic residues of Fd and basic residues of FNR. We have used differential chemical modification of Fd to locate aspartic and glutamic acid residues at the intermolecular interface of the Fd:FNR complex (both proteins from spinach). Carboxyl groups of free and FNR-bound Fd were amidated with carbodiimide/2-aminoethane sulfonic acid (taurine). The differential reactivity of carboxyl groups was assessed by double isotope labeling. Residues protected in the Fd:FNR complex were D-26, E-29, E-30, D-34, D-65, and D-66. The protected residues belong to two domains of negative electrostatic surface potential on either side of the iron-sulfur cluster. The negative end of the molecular dipole moment vector of Fd (377 Debye) is close to the iron-sulfur cluster, in the center of the area demarcated by the protected carboxyl groups. The molecular dipole moment and the asymmetric surface potential may help to orient Fd in the reaction with FNR. In support, we find complementary domains of positive electrostatic potential on either side of the FAD redox center of FNR. The results allow a binding model for the Fd:FNR complex to be constructed.

Comparison of the binding sites of plant ferredoxin for two ferredoxin-dependent enzymes.

Differential chemical modification of acidic residues was used to map the binding site of plant ferredoxin (Fd) for the chloroplast enzyme ferredoxin:thioredoxin reductase (FTR). Binding of FTR to Fd inhibits chemical modification of Fd residues D34, D65, E92, E93, E94 and C-terminal A97. The binding site demarcated by these residues differs from that for ferredoxin:NADP+ reductase (FNR). The FTR site includes C-terminal residues but not helix 24-31, which is part of the FNR site. Both sites enclose the [2Fe-2S] cluster.

Ferredoxin binding site on ferredoxin: NADP+ reductase. Differential chemical modification of free and ferredoxin-bound enzyme.

The chloroplast enzyme ferredoxin: NADP+ reductase (FNR) catalyzes the reduction of NADP+ by ferredoxin (Fd). FNR and Fd form a 1:1 complex that is stabilized by electrostatic interactions between acidic residues of Fd and basic residues of FNR. To localize lysine residues at the Fd binding site of FNR, the FNR:Fd complex (both proteins from spinach) was studied by differential chemical modification. In a first set of experiments, free FNR and the FNR:Fd complex were reacted with the N-hydroxysuccinimidyl ester of biotin. Biotinylated peptides and non-biotinylated peptides were separated on monovalent avidin-Sepharose and purified by high-performance liquid chromatography. Two peptides containing Lys18 and Lys153, respectively, were less biotinylated in complexed FNR than in free FNR. In a second set of experiments, free and complexed FNR were treated with 4-N,N-dimethylaminoazobenzene-4'-isothiocyano-2'-sulfonic acid (S-DABITC) to obtain coloured lysine-modified FNR. Protection of Lys153 was again found by modification with S-DABITC. In addition, Lys33 and Lys35 were less labelled in the S-DABITC-modified. Fd-bound enzyme. FNR modified in the presence, but not in the absence, of Fd was still able to bind Fd, indicating that the Fd-protected residues are involved in the formation of the Fd:FNR complex. The lysine residues disclosed by differential modification surround the positive end of the molecular dipole moment (558 Debye approximately 1.85 x 10(-27) Cm) and are located in a domain of strong positive potential on the surface of the FNR molecule. This domain we had proposed to belong to the binding site of FNR for Fd [De Pascalis, A. R., Jelesarov, I., Ackermann, F., Koppenol, W. H., Hirasawa, M., Knaff, D. B. & Bosshard, H. R. (1993) Protein Science 2. 1126-1135]. The prediction was based on the complementarity of shape between positive and negative potential domains of FNR and Fd, respectively.