Ferredoxin binding site on ferredoxin: NADP⁺ reductase

Abstract

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 Lysl8 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 Lysl53 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 -1.85 X lopz7 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

Anabaena Flavodoxin as an Electron Carrier from Photosystem I to Ferredoxin-NADP+ Reductase. Role of Flavodoxin Residues in Protein−Protein Interaction and Electron Transfer

“…The structures of the FNR-Fd complexes from Anabaena and maize have been determined by X-ray crystallography (9, 10), confirming the importance of the nature of the interactions that was established by biochemical studies. Thus, biochemical and structural data indicate that Fd binds in a concave cavity around the FAD group of the reductase, where residues Lys75, Leu76, and Leu78 on the FNR surface play a crucial role in complex formation, by interacting with the side chains of Glu94 and Phe65 on [11][12][13][14].Flavodoxins (Fld) are small R/ flavoproteins that contain a noncovalently bound FMN cofactor. In cyanobacteria and certain algae, Fld is synthesized instead of Fd when the organism is grown under low-iron conditions and replaces it in the transfer of one electron from PSI to FNR by shuttling between the semiquinone and hydroquinone states (15).…”

“…The structures of the FNR-Fd complexes from Anabaena and maize have been determined by X-ray crystallography (9,10), confirming the importance of the nature of the interactions that was established by biochemical studies. Thus, biochemical and structural data indicate that Fd binds in a concave cavity around the FAD group of the reductase, where residues Lys75, Leu76, and Leu78 on the FNR surface play a crucial role in complex formation, by interacting with the side chains of Glu94 and Phe65 on Fd (5)(6)(7)(8)(9)(11)(12)(13)(14).…”

Crystal Structure of Paprika Ferredoxin-NADP+Reductase

“…In comparison with the Fd-like domain of PDR, the complexed Fd seems to be rotated nearly 90°around a vertical axis running through its iron-sulfur cluster. However, a PDR-like rotation of the complexed Fd would no longer be consistent with the observed cross-linking results (45)(46)(47)(48)(49)(50)(51)(52).…”

“…The third patch is very large, including residues Lys 348 , Lys 349 , Lys 352 , and Lys 353 , while the fourth patch is located around the positively charged residue Lys 323 . Sequence comparison with spinach and Anabaena FNR show that most of these positively charged residues have been implicated in ferredoxin binding in the mentioned biochemical modification studies (45)(46)(47)(48)(49)(50)(51)(52).…”

“…Nevertheless, these models were obtained by manually placing the ironsulfur cluster of Fd to the proposed binding cleft of FNR near the exposed portion of the flavin to account for the results of the biochemical studies (45)(46)(47)(48)(49)(50)(51)(52). Here we present an FNR-Fd docking model generated by computational molecular modeling.…”