NADPH-cytochrome-P-450 reductase promoted hydroxyl radical production by the iron(III)-ochratoxin A complex

The Fe3+ complex of ochratoxin A has been shown to produce hydroxyl radicals in the presence of NADPH and NADPH-cytochrome-P-450 reductase. ESR spin-trapping experiments carried out in the presence of the hydroxyl radical scavenger ethanol and the spin trap DMPO (5,5-dimethyl-1-pyrroline-1-oxide) produced ESR spectra characteristic of the hydroxyl radial-derived carbon-centered DMPO-alkoxyl radical adduct. Thus hydroxyl radicals produced by the Fe3(+)-ochratoxin A complex in the presence of an enzymatic reductase may be be partly responsible for ochratoxin A toxicity.     

Docking analysis of transient complexes: interaction of ferredoxin-NADP+ reductase with ferredoxin and flavodoxin

Ferredoxin (Fd) interacts with ferredoxin-NADP(+) reductase (FNR) to transfer two electrons to the latter, one by one, which will finally be used to reduce NADP(+) to NADPH. The formation of a transient complex between Fd and FNR is required for the electron transfer (ET), and extensive mutational and crystallographic studies have been reported to characterize such protein-protein interaction. However, some aspects of the association mechanism still remain unclear. Moreover, in spite of their structural differences, flavodoxin (Fld) can replace Fd in its function and interact with FNR to transfer electrons with only slightly lower efficiency. Although crystallographic structures for the FNR:Fd association have been reported, experimental structural data for the FNR:Fld interaction are highly elusive. We have modeled here the interactions between FNR and both of its protein partners, Fd and Fld, using surface energy analysis, computational rigid-body docking simulations, and interface side-chain refinement. The results, consistent with previous experimental data, suggest the existence of alternative binding modes in these ET proteins.     

Absorbance spectral change of the cytochrome P-450S21-phenylisocyanide complex upon binding of reduced NADPH-cytochrome-P-450 reductase.

Reduction of cytochrome P-450S21 (SF) (SF, substrate-free; purified from bovine adrenocortical microsomes) with sodium dithionite (Na2S2O4) in the presence of phenylisocyanide produced a ferrous cytochrome P-450S21 (SF)-phenylisocyanide complex with Soret absorbance maxima at 429 and 456 nm. On the other hand, when a preformed ferric cytochrome P-450S21 (SF)-NADPH-cytochrome-P-450 reductase (Fp2) complex was reduced chemically or enzymatically under the same conditions, the absorbance spectrum of the ferrous cytochrome P-450S21 (SF)-phenylisocyanide complex changed drastically, as characterized by an increase in absorbance intensity at 429 nm and a decrease at 456 nm. Similar spectral changes were observed by addition of reduced Fp2 to the preformed ferrous cytochrome P-450S21 (SF)-phenylisocyanide complex. Experiments to reduce a ferric cytochrome P-450S21 (SF)-phenylisocyanide complex with sodium dithionite in the presence of various amounts of Fp2 showed that; (1), the spectral change reached maxima for both absorption increase at 429 nm and decrease at 456 nm when cytochrome P-450S21 and Fp2 were previously mixed at the cytochrome P-450S21:Fp2 ratio of 1:5; (2), the spectral change was suppressed in 300 mM potassium phosphate buffer (pH 7.4). These results suggest that the absorbance spectral change is due to a conformational change around the heme moiety induced by association with reduced Fp2.     

Biochemical characterization of ferredoxin-NADP(+) reductase interaction with flavodoxin in Pseudomonas putida

Flavodoxin (Fld) has been demonstrated to bind to ferredoxin- NADP(+) reductase A (FprA) in Pseudomonas putida. Two residues (Phe(256), Lys(259)) of FprA are likely to be important for interacting with Fld based on homology modeling. Sitedirected mutagenesis and pH-dependent enzyme kinetics were performed to further examine the role of these residues. The catalytic efficiencies of FprA-Ala(259) and FprA-Asp(259) proteins were two-fold lower than those of the wild-type FprA. Homology modeling also strongly suggested that these two residues are important for electron transfer. Thermodynamic properties such as entropy, enthalpy, and heat capacity changes of FprA-Ala(259) and FprA-Asp(259) were examined by isothermal titration calorimetry. We demonstrated, for the first time, that Phe(256) and Lys(259) are critical residues for the interaction between FprA and Fld. Van der Waals interactions and hydrogen bonding were also more important than ionic interactions for forming the FprA-Fld complex. [BMB Reports 2012; 45(8): 476-481].     

Chemical modification of the active site of ferredoxin-NADP reductase and conformation of the binary ferredoxin/ferredoxin-NADP reductase complex in solution

Ferredoxin-NADP⁺ reductase (EC 1.18.1.2) was chemically modified by the triplet probe eosin isothiocyanate (eosin-NES). Incorporation of 1 mol eosin-NCS/mol ferredoxin-NADP⁺ reductase completely inhibited binding of NADP⁺/NADPH to the enzyme. Binding of eosin without the reactive group to the enzyme was shown to be reversible but to compete with NADP⁺/NADPH with a K_i of approx. 5 μM. The binding site of eosin-NCS has been located in the primary sequence ferredoxin-NADP⁺ reductase. After specific cleavage of arginine with trypsin a single labelled peptide was obtained and identified as the fragment from residue 179–228 in the primary sequence. Binding of eosin-NCS occurred in either of two predicted helices (residues 179–189 or 212–228) which are both part of an /β structure characteristic for nucleotide binding folds. The rotational diffusion in solution of the eosin-labelled ferredoxin-NADP⁺ reductase and its complex with ferredoxin was measured with laser flash spectroscopy under photoselection. From the measured rotational correlation times and the known structure of ferredoxin-NADP⁺ reductase at 3.7 Å resolution, we propose that ferredoxin is bound to ferredoxin-NADP⁺ reductase between the two domains of the flavoprotein. The two ferredoxin-NADP⁺ reductase domains and ferredoxin form a triangle which results in a highly integrated binary complex.     

Oxidation-reduction potentials of ferredoxin-NADP+ reductase and flavodoxin from Anabaena PCC 7119 and their electrostatic and covalent complexes.

The oxidation-reduction potentials of ferredoxin-NADP+ reductase and flavodoxin from the cyanobacterium Anabaena PCC 7119 were determined by potentiometry. The potentials at pH 7 for the oxidized flavodoxin/flavodoxin semiquinone couple (E2) and the flavodoxin semiquinone/hydroquinone couple (E1) were -212 mV and -436 mV, respectively. E1 was independent of pH above about pH 7, but changed by approximately -60 mV/pH below about pH 6, suggesting that the fully reduced protein has a redox-linked pKa at about 6.1, similar to those of certain other flavodoxins. E2 varied by -50 mV/pH in the range pH 5-8. The redox potential for the two-electron reduction of ferredoxin-NADP+ reductase was -344 mV at pH 7 (delta Em = -30 mV/pH). In the 1:1 electrostatic complex of the two proteins titrated at pH 7, E2 was shifted by +8 mV and E1 was shifted by -25 mV; the shift in potential for the reductase was +4 mV. The potentials again shifted following treatment of the electrostatic complex with a carbodiimide, to covalently link the two proteins. By comparison with the separate proteins at pH 7, E2 for flavodoxin shifted by -21 mV and E1 shifted by +20 mV; the reductase potential shifted by +2 mV. The potentials of the proteins in the electrostatic and covalent complexes showed similar pH dependencies to those of the individual proteins. Qualitatively similar changes occurred when ferredoxin-NADP+ reductase from Anabaena variabilis was complexed with flavodoxin from Azotobacter vinelandii. The shifts in redox potential for the complexes were used with previously determined values for the dissociation constant (Kd) of the electrostatic complex of the two oxidised proteins, in order to estimate Kd values for the interaction of the different redox forms of the proteins. The calculations showed that the electrostatic complexes, formed when the proteins differ in their redox states, are stronger than those formed when both proteins are fully oxidized or fully reduced.     

Effects of chemical modification of Anabaena flavodoxin and ferredoxin-NADP+ reductase on the kinetics of interprotein electron transfer reactions.

The influence of chemical modification of arginine residues (using phenylglyoxal) in ferredoxin-NADP+ reductase (FNR), and of carboxyl groups (using glycine ethyl ester) in flavodoxin (Fld), on the kinetics of electron transfer between FNR and Fld, and between ferredoxin (Fd) and FNR, was examined using laser flash photolysis methods. All proteins were obtained from the cyanobacterium Anabaena PCC7119. Reduction by laser-generated 5-deazariboflavin semiquinone of the FAD moiety of phenylglyoxal-modified FNR occurred with a second-order rate constant 2.5-fold smaller than that obtained for reduction of native FNR, indicating either a small degree of steric hindrance of the cofactor, or a decrease in its redox potential, upon chemical modification. In contrast, no changes were found in the kinetics of reduction of the FMN cofactor of Fld modified by glycine ethyl ester as compared with the native protein. The observed rate constants for reoxidation of Fdred (reduced Fd) by FNRox (oxidized FNR) were dramatically decreased (approximately 100-fold) when phenylglyoxal-modified FNR was used. In contrast to the reaction involving the native proteins, no ionic strength effects on kobs values were found. These results, and those obtained upon varying the protein concentration, indicate that the rate constant for complex formation and the attractive electrostatic interaction between the two proteins were greatly diminished by chemical modification of arginine residues of FNR. When phenylglyoxal-modified FNRsq (FNR semiquinone) was used to reduce Fldox (oxidized Fld), similar inhibitory effects were observed. In this case, the limiting first-order rate constant for Fldsq (Fld semiquinone) formation via intracomplex electron transfer from FNRsq was approximately 12-fold smaller than that obtained for the native FNR (600 s-1 vs 7000 s-1). Again, ionic strength effects were diminished. The glycine-ethyl-ester-modified Fld yielded a limiting first-order rate constant for intracomplex electron transfer from FNRsq to Fldox which was approximately 7-fold smaller (1000 s-1) than that obtained with native Fld, and ionic strength effects were again diminished. These results indicate that complex formation can still occur between modified FNR and native Fld, and between native FNR and modified Fld, but that the geometry of these complexes is altered so as to decrease the effectiveness of interprotein electron transfer. The results are discussed in terms of the specific structural features of the proteins involved.     

Anabaena flavodoxin as an electron carrier from photosystem I to ferredoxin-NADP+ reductase. Role of flavodoxin residues in protein-protein interaction and electron transfer

Biochemical and structural studies indicate that electrostatic and hydrophobic interactions are critical in the formation of optimal complexes for efficient electron transfer (ET) between ferredoxin-NADP(+) reductase (FNR) and ferredoxin (Fd). Moreover, it has been shown that several charged and hydrophobic residues on the FNR surface are also critical for the interaction with flavodoxin (Fld), although, so far, no key residue on the Fld surface has been found to be the counterpart of such FNR side chains. In this study, negatively charged side chains on the Fld surface have been individually modified, either by the introduction of positive charges or by their neutralization. Our results indicate that although Glu16, Glu20, Glu61, Asp65, and Asp96 contribute to the orientation and optimization of the Fld interaction, either with FNR or with photosystem I (PSI) (presumably through the formation of salt bridges), for efficient ET, none of these side chains is involved in the formation of crucial salt bridges for optimal interaction with FNR. These data support the idea that the FNR-Fld interaction is less specific than the FNR-Fd interaction. However, analysis of the reactivity of these mutated Flds toward the membrane-anchored PSI complex indicated that all mutants, except Glu16Gln, lack the ability to form a stable complex with PSI. Thr12, Thr56, Asn58, and Asn97 are present in the close environment of the isoalloxazine ring of FMN in Anabaena Fld. Their roles in the interaction with and ET to FNR and PSI have also been studied. Mutants at these Fld positions indicate that residues in the close environment of the isoalloxazine ring modulate the ability of Fld to bind to and to exchange electrons with its physiological counterparts.     

Preparation and properties of a cross-linked complex between ferredoxin--NADP+ reductase and flavodoxin

The electrostatically stabilized complex between Anabaena variabilis ferredoxin--NADP+ reductase and Azotobacter vinelandii flavodoxin has been covalently cross-linked by treatment with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide. The covalent complex exhibits a molecular mass and FMN/FAD content consistent with that expected for a 1:1 stoichiometry of the two flavoproteins. Immunochemical cross-reactivity is exhibited by the covalent complex with rabbit antisera prepared separately against each protein. The complex retains NADPH-ferricyanide diaphorase activity although the Km for ferricyanide is increased twofold and the turnover number is decreased by a factor of two when compared to native reductase. NADPH-cytochrome-c reductase activity of the complex is observed at a level that is quite similar to that determined at saturating concentrations of flavodoxin, while it is only 1-2% of that exhibited by the reductase in the presence of ferredoxin. No stimulation of cytochrome-c reductase activity is observed on adding ferredoxin to the cross-linked complex. Stopped-flow data show that covalent cross-linking of the flavodoxin to the reductase reduces the rate of electron transfer from its semiquinone form to cytochrome c by a factor of 60. Anaerobic titrations of the reduced complex with NADP+ show the semiquinone/quinol couple of the flavodoxin is increased 100 mV relative to the free form and the quinone/quinol couple of complexed ferredoxin-NADP+ reductase is increased by only 25 mV, relative to the free protein. Addition of NADPH to the cross-linked complex reduces the FAD of the reductase as well as the FMN moiety of flavodoxin to a mixture of semiquinone and quinol forms.     

Purification of ferredoxin-NADP+ reductase, flavodoxin and ferredoxin from a single batch of the cyanobacterium Anabaena PCC 7119.

Methods are described for the simultaneous isolation of ferredoxin-NADP+ reductase, ferredoxin and flavodoxin from large quantities of the cyanobacterium Anabaena PCC 7119 allowing the use of a single batch of cells. The ultraviolet-visible spectra and the extinction coefficients of ferredoxin-NADP+ reductase and ferredoxin were determined. The purification procedure also yields enriched fractions of phycobiliproteins and cytochrome c553.     

Resolution and reconstitution of spinach ferredoxin-NADP+ reductase

The apoprotein from spinach ferredoxin-NADP⁺ reductase was prepared by treatment with 3 M calcium chloride. This procedure caused complete removal of the FAD prosthetic group together with considerable denaturation of the apoprotein. Thus, the recovery of total activity upon reconstitution with FAD was only 30%. More importantly, however, both transhydrogenase and diaphorase activities were 70% of that native enzyme based on bound flavin. The visible spectrum and properties of the reconstituted reductase were undiscernible from those of the native protein.     

31P-nuclear-magnetic-resonance study of muscles from Mytilus edulis

Phosphorus-31 nuclear magnetic resonance (NMR) spectra were recorded from freshly excised and resting anterior byssus retractor muscles of the mussel Mytilus edulis. The absolute concentrations of phosphometabolites measured by NMR compare well with the values obtained by a biochemical method. Quantitative measurements were achieved in several minutes by applying to the observed NMR signals a correction taking the saturation effects into account. The time evolution of the phosphometabolite concentrations reveals that the hydrolysis of phosphoarginine is a first-order reaction producing inorganic phosphate, whilst the adenosine triphosphate level remains constant for more than 10 h. The phosphoarginine hydrolysis rate varies as a function of the season and reaches a maximum during the reproduction period of the mussel. This increase in phosphoarginine consumption could be bound up with the higher excitability of the muscles during spring. The activation energy of the phosphoarginine hydrolysis reaction also depends on the season and the difference between the value determined in winter and that measured in spring is explained by a modification of the mode of action or of the proportions of the enzymes involved in the muscular metabolism.     

Role of hydrophobic interactions in the flavodoxin mediated electron transfer from photosystem I to ferredoxin-NADP+ reductase in Anabaena PCC 7119

Hydrophobic interactions play an active role in effective complex formation between ferredoxin-NADP(+) reductase (FNR) and ferredoxin (Fd) from Anabaena, where an aromatic amino acid residue on the Fd surface (F65) and three hydrophobic residues (L76, L78, and V136) on the reductase surface have been shown to be essential for the efficient electron transfer (ET) reaction between Fd and FNR (Martínez-Júlvez et al. (2001) J. Biol. Chem. 276, 27498-27510). Since in this system flavodoxin (Fld) can efficiently replace Fd in the overall ET process, we have further investigated if such hydrophobic interactions are also critical in complex stabilization and ET in the FNR/Fld association. Different ET behaviors with Fld are observed for some of the mutations made at L76, L78, and V136 of Anabaena FNR. Thus, the ET interaction with Fld is almost completely lost upon introduction of negatively charged side chains at these positions, while more conservative changes in the hydrophobic patch can influence the rates of ET to and from Fld by altering the binding constants and the midpoint redox potentials of the flavin group. Therefore, our results confirm that nonpolar residues in the region close to the FAD group in FNR participate in the establishment of interactions with Fld, which serve to orient the two flavin groups in a manner such that ET is favored. In an attempt to look for the counterpart region of the Fld surface, the effect produced by the replacement of the only two nonpolar residues on the Fld surface, I59 and I92, by a Lys has also been analyzed. The results obtained suggest that these two hydrophobic residues are not critical in the interaction and ET processes with FNR. The reactivity of these I92 and I59 Fld mutants toward the membrane-anchored photosystem I (PSI) complex was also analyzed by laser flash absorption spectroscopy. From these data, significant effects are evident, especially for the I92 position of Fld, both in the association constant for complex formation and in the electron-transfer rate constant in the PSI/Fld system.     

Crystals of Anabaena PCC 7119 ferredoxin-NADP+ reductase

Crystals of ferredoxin-NADP+ reductase (FNR) from the cyanobacterium Anabaena PCC 7119 are grown in the presence of polyethylene glycol 6000 and beta-octyl glucoside. They belong to the hexagonal system. The cell parameters are a = b = 87.8 A, c = 92.7 A, space group P6(1) or P6(5), and a Vm of 3.0 A3/dalton for one molecule of 36,000 daltons per asymmetric unit. These crystals diffract strongly up to 1.9 A and are suitable for X-ray structural studies.     

Identification of specific carboxyl groups on Anabaena PCC 7119 flavodoxin which are involved in the interaction with ferredoxin-NADP+ reductase.

Flavodoxin from the nitrogen-fixing cyanobacteria Anabaena PCC 7119 forms an electron-transfer complex with ferredoxin--NADP+ reductase (FNR) from the same organism. The complex is mainly governed by electrostatic interactions between side-chain amino groups of the reductase and carboxyl residues of flavodoxin. In order to localize the binding site on flavodoxin, chemical modification of its carboxyl groups has been carried out. Treatment of flavodoxin with a water-soluble carbodiimide, N-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), in the presence of a nucleophile, glycine ethyl ester, caused a time-dependent modification of the protein that is responsible for the loss of its ability to participate as electron carrier in the photoreduction of NADP+ by chloroplast membranes, and also in NADPH--cytochrome-c reductase activity, by about 85%. Nevertheless, the ability of flavodoxin to receive electrons from the reducing side of photosystem I was much less affected. The inhibition was enhanced at low pH, suggesting that carboxylic acid groups were the target of chemical modification. Treated flavodoxin failed to form covalent complexes with FNR and the dissociation constant for the non-covalent complex with FNR was fourfold higher. After tryptic digestion of a sample of flavodoxin modified by EDC in the presence of [1-14C]glycine ethyl ester, two major radioactive peptides were isolated. The first protein fragment contained three carboxylic residues (Asp123, Asp126 and Asp129), corresponding to the region where long-chain flavodoxins show an insert compared to short-chain flavodoxins. The second peptide corresponded to a similar region, either in the amino acid sequence or in the three-dimensional structure of the protein and also containing three carboxyl groups (Asp144, Glu145 and Asp146). Four of these carboxyl groups (Asp123, Asp126, Asp144 and Asp146) are highly conserved in all long-chain flavodoxins, suggesting that they could play an essential role in substrate recognition.     

A 31P-nuclear-magnetic-resonance study of the phosphate groups in lipopolysaccharide and lipid A from Salmonella

Untreated and partially deacylated lipopolysaccharides from various P- and P+ strains of Salmonella were studied with 31P nuclear magnetic resonance spectroscopy and by conventional analytical methods. The spectral signals were assigned to various phosphate groups in the lipid A moiety and in the oligosaccharide part. A signal at +2.3 ppm could be assigned to a phosphodiester linkage formed between 4-amino-4-deoxyl-L-arabinose linked via the glycosidic hydroxyl group to the 4'-phosphate group of the glucosamine disaccharide in the lipid A moiety. A strong pyrophosphate signal at +11 ppm in P- strains was identified as a pyrophosphoryl ethanolamine group at the glycosidic end of this glucosamine disaccharide unit. No evidence was found for phosphodiester or pyrophosphodiester bonds crosslinking lipopolysaccharide 'subunits'. A revised version of the lipid A structure of Salmonella is presented. By a combination of 31P nuclear magnetic resonance spectroscopy data and conventional analytical methods the extent to which the lipopolysaccharides are substituted by various phosphate groups on the lipid A and the oligosaccharide moiety could be estimated. It was thus shown that substantial heterogeneity, leading to several molecular species of lipopolysaccharides is caused by addition or omission of certain groups. Since changes in substitution were found to be dependent on the growth conditions, it is thought possible that the overall negative surface charge of Salmonella can be modified by addition or omission of neutralising amino groups from ethanolamine and/or 4-amino-4-deoxy-L-arabinose, and can thus be adapted to the environment.     

Circular dichroism studies of the complex between ferredoxin and ferredoxin-NADP reductase

Circular dichroism (CD) spectra are presented of ferredoxin, ferredoxin-NADP reductase and their complex. A change in CD occurs on complex formation which is consistent with a decrease in the Cotton effects due to the ferredoxin. This change is interpreted as due to a decrease in interaction in ferredoxin between the iron-sulphur chromophore group and the protein.     

Flavodoxin-mediated electron transfer from photosystem I to ferredoxin-NADP+ reductase in Anabaena: role of flavodoxin hydrophobic residues in protein-protein interactions

Three surface hydrophobic residues located at the Anabaena flavodoxin (Fld) putative complex interface with its redox partners were replaced by site-directed mutagenesis. The effects of these replacements on Fld interaction with both its physiological electron donor, photosystem I (PSI), and its electron acceptor, ferredoxin-NADP+ reductase (FNR), were analyzed. Trp57, Ile59, and Ile92 contributed to the optimal orientation and tightening of the FNR:Fld and PSI:Fld complexes. However, these side chains did not appear to be involved in crucial specific interactions, but rather contributed to the obtainment of the optimal orientation and distance of the redox centers required for efficient electron transfer. This supports the idea that the interaction of Fld with its partners is less specific than that of ferredoxin and that more than one orientation is efficient for electron transfer in these transient complexes. Additionally, for some of the analyzed processes, WT Fld seems not to be the most optimized molecular species. Therefore, subtle changes at the isoalloxazine environment not only influence the Fld binding abilities, but also modulate the electron exchange processes by producing different orientations and distances between the redox centers. Finally, the weaker apoflavodoxin interaction with FNR suggests that the solvent-accessible region of FMN plays a role either in complex formation with FNR or in providing the adequate conformation of the FNR binding region in Fld.     

Comparison of the kinetics of reduction and intramolecular electron transfer in electrostatic and covalent complexes of ferredoxin-NADP+ reductase and flavodoxin from Anabaena PCC 7119

The kinetics of reduction and intracomplex electron transfer in electrostatically stabilized and covalently crosslinked complexes between ferredoxin-NADP+ reductase (FNR) and flavodoxin (Fld) from the cyanobacterium Anabaena PCC 7119 were compared using laser flash photolysis. The second-order rate constant for reduction by 5-deazariboflavin semiquinone (dRfH) of FNR within the electrostatically stabilized complex at 10 mM ionic strength (4.0 X 10(8) M-1 s-1) was identical to that for free FNR. This suggests that the FAD cofactor of FNR is not sterically hindered upon complex formation. A lower limit of approximately 7000 s-1 was estimated for the first-order rate constant for intracomplex electron transfer from FNRred to Fldox under these conditions. In contrast, for the covalently crosslinked complex, a smaller second-order rate constant (2.1 X 10(8) M-1 s-1) was obtained for the reduction of FNR by dRfH within the complex, suggesting that some steric hindrance of the FAD cofactor of FNR occurs due to crosslinking. A limiting rate constant of 1000 s-1 for the intracomplex electron transfer reaction was obtained for the covalent complex, which was unaffected by changes in ionic strength. The substantially diminished limiting rate constant, relative to that of the electrostatic complex, may reflect either a suboptimal orientation of the redox cofactors within the covalent complex or a required structural reorganization preceding electron transfer which is not allowed once the proteins have been covalently linked. Thus, although the covalent complex is biochemically competent, it is not a quantitatively precise model for the catalytically relevant intermediate along the reaction pathway.     

Molecular heterogeneity of ferredoxin-NADP+ reductase from spinach leaves

Highly purified ferredoxin-NADP+ reductase from spinach leaves showed at least eight different protein bands in the electrofocused gel. All of them were catalytically active and were adsorbed on a ferredoxin-Sepharose 4B affinity column. The N-terminal amino acid sequence of the main component species was analyzed by the automatic Edman degradation method. It was found that when the reductase was stored at 4 degrees C, new protein bands appeared in isoelectric focusing and sodium dodecyl sulfate polyacrylamide gel electrophoreses, but the appearance of the bands was suppressed by the addition of a protease inhibitor, diisopropyl fluorophosphate. This indicates that the molecular heterogeneity of the reductase may result from the digestion with a protease present in spinach leaves.