The NADP⁺‐binding site of ferredoxin‐NADP⁺ reductase

Abstract

The flavoprotein ferredoxin-NADP' reductase is inactivated and loses its ability to bind NADP' during covalent modification of a lysine by 5-dimethylaminonaphthalene-I-sulfonyl chloride (dansyl chloride) [Zanetti, G. (1976) Biochim. Biophys. Acta 445, [14][15][16][17][18][19][20][21][22][23][24]. The substrate NADP' gives almost complete protection against inactivation and modification. These observations are extended in this report by the characterization of an octapeptide containing the dansyl-lysine which was isolated by high-performance liquid chromatography from tryptic digests of protein modified with radiolabeled reagent. The amount of this peptide was severely reduced in protein modified in the presence of NADP'. The sequence of the dansyl-peptide, only partially obtained by Edman degradation, was completed by analysis of the fragments resulting from thermolysin digestion of the purified tryptic dansyl-peptide. Thus, the octapeptide containing the essential lysine residue has the following [6] residues are located in the NADP+-binding site of the reductase. With the aim to further characterize this region of the protein, we decided to isolate the peptide containing the essential lysine residue [3]. Chemical modification with dansyl chloride, yielding a covalent fluorescent group attached to the residue, facilitates the recognition and the isolation of such a peptide. Furthermore, the selectivity of this labeling was well established [3], since a differential incorporation of only 1 -1.5 mol dansyl/mol FAD was found between free and NADP+-protected enzyme. Here we report the sequence of an octapeptide thus isolated. A comparison is made with other NADP+-dependent dehydrogenases.

Lysine residues on ferredoxin‐NADP+ reductase from Anabaena sp. PCC 7119 involved in substrate binding

“…The peptide which displayed the highest degree of fluorescence (4 in Fig. 1 Fd and spinach FNR [3], as was previously predicted by chemical modification studies in the spinach enzyme [7,8,18]. …”

Identification of Lys116 as the target of N‐ethylmaleimide inactivation of ferredoxin:NADP+ oxidoreductase

“…This residue was already identified as the target of dansyl chloride inactivation of the enzyme [5]. Protection afforded by NADP(H) against modification and the fact that the reductive half-reaction of the enzyme became impaired (same inactivation rate for diaphorase or ferredoxindependent reductase activities and lack of protection by the presence of ferredoxin in the incubation mixture) indicate that Lysl16 is involved in pyridine-nucleotide binding.…”

“…Here we report that FNR inactivation resulted from modification of Lysll6, the same residue which was responsible for dansyl chloride inactivation [5]. No sulfhydryl groups were found to be alkylated.…”

Analysis of cDNA clones encoding the entire precursor-polypeptide for ferredoxin: NADP+ oxidoreductase from spinach

“…While N-and C-terminal regions of the molecule have been assigned to the FAD-and NADPbinding domains, respectively, the relatively low level of resolution attained, probably caused by the existence of polypeptide variants, has precluded disclosure of further meaningful detail (Sheriff and Herriott 1981). However, discrete basic residues, especially Lys 85/88 and Lys 116, have recently been deduced from cross-linking experiments to interact with C-terminal acidic residues of ferredoxin, and to bind the 2' phosphate of NADP +, respectively (Cidaria et al 1985;Merati and Zanetti 1987;Zanetti and Merati 1987;Zanetti et al 1988).…”