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.
Jelesarov
I
I
Biochemisches Institut, Universität Zürich, Switzerland.
De Pascalis
A R
AR
Koppenol
W H
WH
Hirasawa
M
M
Knaff
D B
DB
Bosshard
H R
HR
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D001665:Binding Sites; D001710:Biotin; D002851:Chromatography, High Pressure Liquid; D005287:Ferredoxin-NADP Reductase / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D005288:Ferredoxins / Q000378:metabolism*; D017879:Isothiocyanates; D008239:Lysine / Q000032:analysis* / Q000378:metabolism; D008969:Molecular Sequence Data; D009994:Osmolar Concentration; D010084:Oxidation-Reduction; D013861:Thiocyanates; D014675:Vegetables / Q000201:enzymology; D004124:p-Dimethylaminoazobenzene / Q000031:analogs & derivatives
D000596:Amino Acids; D005288:Ferredoxins; D017879:Isothiocyanates; D013861:Thiocyanates; D001710:Biotin; C012926:4-(N,N-dimethylaminoazobenzene)-4'-isothiocyanate; D004124:p-Dimethylaminoazobenzene; D005287:Ferredoxin-NADP Reductase; D008239:Lysine