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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.

1. Ferredoxin-NADP' reductase resolved into apoprotein and flavin by incubation with 2.5 M CaClz at pH 7.5 and 2 ^C. Essential factors to recover a reconstitutable apoprotein are dithiothreitol, glycerol and guanidine/HCI. The apoprotein is stable for at least a week at -20 "C.2. The release of the prosthetic group from the protein by the CaZ + ions is a multi-step process. Three different effects of these ions are identifiable: (a) a rapid 20-25% inhibition of catalytic activity, probably caused by an increase in the ionic strength of the medium; other cations can produce it as well; (b) a slower induction of a conformational change in the protein which causes complete loss of activity and exposure to solvent of the flavin moiety; the F A D is finally released from the protein; (c) complete conversion of F A D to FMN, which blocks reconstitution to holoenzyme, caused by the well-known hydrolytic action of Ca2' ions on the pyrophosphate bridge of F A D 3. Binding of F A D by the apoferredoxin-NADP' reductase is very rapid and it is complete in a few minutes even at 0 "C. A Kd of 3.4 x M has been determined by fluorescence titration. The reconstituted holoenzyme has catalytic activity, spectral and fluorescence properties nearly identical to the native enzyme. The gel electrophoresis and isoelectrofocusing patterns of the two enzymes are very similar. Removal of factors from the apoprotein solution such as dithiothreitol and glycerol promotes the appearance of protein aggregates.

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