Reexamination of the interaction between ferredoxin:NADP reductase and NADP

A comparison was made of graphical and subtractive methods for the determination of the dissociation constant of a complex between ferredoxin:NADP reductase and NADP. The subtractive method gave K_d values near 10 μm which are consistent with recently determined values for K_m,NADP in assays of NADP photoreduction by chloroplast membranes. The graphical method gave values which were considerably higher. The difference between the two methods is due to the failure of the graphical method to correct for the amount of each component present in the complex at the low NADP/ flavoprotein ratios necessary for binding studies. A second NADP binding site of much lower affinity (K_d approx 1 mm) was also detected.     

Inhibition of adrenodoxin reductase by NADP+ and NADPH

Adrenodoxin reductase (EC 1.18.1.2) catalyzes the oxidation of NADPH by 1.4-benzoquinone. The catalytic constant of this reaction at pH 7.0 is equal to 25-28 s-1. NADP+ acts as the mixed-type nonlinear inhibitor of enzyme increasing Km of NADPH and decreasing catalytic constant. NADP+ and NADPH act as mutually exclusive inhibitors relative to reduced adrenodoxin reductase. The patterns of 2',5'-ADP inhibition are analogous to that of NADP+. These data support the conclusion about the existence of second nicotinamide coenzyme binding centre in adrenodoxin reductase.     

Effect of cadmium on ferredoxin:NADP+ oxidoreductase activity

Ferredoxin:NADP(+) oxidoreductase (FNR) was treated with cadmium and after that its diaphorase reaction in the presence of dibromothymoquinone (DBMIB) or ferricyanide (FeCy, K(3)Fe(CN)(6)) was examined. CdSO(4) (5 mM) caused 50% inhibition after half hour incubation. At least two components were distinguishable in the time-course inhibition, suggesting that more than one amino acid residues were engaged in reaction with the metal ion. The Lineweaver-Burk plots indicate that Cd(2+) is an uncompetitive inhibitor for DBMIB reduction but exerts non-competitive inhibition for the NADPH oxidation. The FeCy reduction did not follow Michaelis-Menten kinetics. Zn(2+) diminished inhibitory effect of Cd(2+) on the DBMIB reduction but enhanced inhibition of the FeCy reduction. Incubation with additional chelator (beta-mercaptoethanol, or histidine) abolished inhibitory effect of Cd(2+) on the FeCy reduction but not on the DBMIB reduction. The mode of Cd(2+) action on the diaphorase activity of FNR in the presence of DBMIB or FeCy is briefly discussed with the special reference to the implication of two distinct sites at the FNR molecule, which might be involved in the reduction of various non-physiological substrates.     

The apicomplexan Cryptosporidium parvum possesses a single mitochondrial-type ferredoxin and ferredoxin:NADP+ reductase system

We have successfully expressed recombinant mitochondrial‐type ferredoxin (mtFd) and ferredoxin:NADP⁺ reductase (mtFNR) from Cryptosporidium parvum and characterized their biochemical features for the first time for an apicomplexan. Both C. parvum mtFd (CpmtFd) and FNR (CpmtFNR) were obtained and purified as holo‐proteins, in which the correct assembly of [2Fe–2S] cluster in Fd and that of FAD in FNR were confirmed and characterized by UV/vis and electron paramagnetic resonance. These proteins were fully functional and CpmtFNR was capable of transferring electrons from NADPH to CpmtFd in a cytochrome c‐coupled assay that followed a typical Michaelis‐Menten kinetics. Apicomplexan mtFd and mtFNR proteins were evolutionarily divergent from their counterparts in humans and animals and could be explored as potential drug targets in Cryptosporidium and other apicomplexans.     

Interaction of ferredoxin with ferredoxin:NADP reductase: effects of chemical modification of ferredoxin

Chemical modification studies have been conducted on spinach ferredoxin to determine the nature of the groups on ferredoxin involved in its interaction with its reaction partners. Modification of a limited number (three or four) carboxyl groups or of the single histidine residue resulted in a decreased ability of ferredoxin to participate in NADP photoreduction but not in cytochrome c photoreduction, suggesting that these groups may be involved in interaction with ferredoxin:NADP reductase but are not involved in interaction with the reducing side of Photosystem I. In contrast, modification of amino groups or the single arginine residue on ferredoxin had little effect on the ability of ferredoxin to participate in NADP photoreduction, suggesting these groups are not involved in the interaction of ferredoxin with either ferredoxin:NADP reductase or the reducing side of Photosystem I. Attempts to modify tyrosine residues on ferredoxin resulted in destruction of the iron-sulfur center of the protein.     

Complex-forming properties of butanedione-modified ferredoxin-NADP+ reductase with NADP+ and ferredoxin.

The plastidic ferredoxin-NADP⁺ reductase from the xanthophycean alga Bumilleriopsis forms a stoichiometric 1:1 complex with ferredoxin and NADP⁺ which is demonstrated by difference spectra of both complexes. Butanedione modification of the flavoprotein results in loss of its enzymatic activities (transhydrogenase and diaphorase) concurrently with its capability to form a complex with NADP⁺, whereas the ferredoxin-binding site is practically not influenced by the modifying reagent and complex formation is still possible. It is assumed, therefore, that butanedione specifically reacts with the arginine residue of the protein involved in binding of pyridine nucleotides at the active site. Further, the data presented strongly support the previous proposal of different binding sites for ferredoxin and pyridine nucleotides at the reductase.     

Characterization of a covalently linked complex involving ferredoxin and ferredoxin: NADP reductase

Ferredoxin and the flavoprotein, ferredoxin: NADP reductase, have been covalently linked by incubation in the presence of a water soluble carbodiimide. The cross-linking reaction yields an adduct having a 1:1 stoichiometry. The adduct has depressed levels of diaphorase and NADPH oxidase activity and is inactive in reduction of cytochrome c using NADPH as an electron donor. Thus, although similar to an adduct described by Zanetti and coworkers [J Biol Chem 259: 6153–6157 (1984)] in its stoichiometry, the adduct described herein has significantly different enzymatic properties. It is suggested that this may be a reflection of differences in the interaction between the two proteins resulting from differences in experimental conditions in which the two adducts were prepared.Abbreviations Fd ferredoxin - Fp ferredoxin: NADP reductase - Fd Fp covalently linked Fd-Fp adduct - Fd:Fp noncovalently linked complex between Fd and Fp - EDC 1-ethyl-3-(dimethylaminopropyl) carbodiimide - Tris tris-hydroxymethylaminomethane - MOPS 3-(N-morpholino)propane sulfonic acid - DCIP 2,6-dichloropenolindophenol     

Electron transfer by ferredoxin:NADP+ reductase. Rapid-reaction evidence for participation of a ternary complex

Rapid reaction studies presented herein show that ferredoxin:NADP+ oxidoreductase (FNR, EC 1.18.1.2) catalyzes electron transfer from spinach ferredoxin (Fd) to NADP+ via a ternary complex, Fd X FNR X NADP+. In the absence of NADP+, reduction of ferredoxin:NADP+ reductase by Fd was much slower than the catalytic rate: 37-80 s-1 versus at least 445 e-s-1; dissociation of oxidized spinach ferredoxin (Fdox) from one-electron reduced ferredoxin:NADP+ reductase (FNRsq) limited the reduction of FNR. This confirms the steady-state kinetic analysis of Masaki et al. (Masaki, R., Yoshikaya, S., and Matsubara, H. (1982) Biochim. Biophys. Acta 700, 101-109). Occupation of the NADP+ binding site of FNR by NADP+ or by 2',5'-ADP (a nonreducible NADP+ analogue) greatly increased the rate of electron transfer from Fd to FNR, releiving inhibition by Fdox. NADP+ (and 2',5'-ADP) probably facilitate the dissociation of Fdox; equilibrium studies have shown that nucleotide binding decreases the association of Fd with FNR (Batie, C. J. (1983) Ph.D. dissertation, Duke University; Batie, C. J., and Kamin, H. (1982) in Flavins and Flavoproteins VII (Massey, V., and Williams, C. H., Jr., eds) pp. 679-683, Elsevier, New York; Batie, C.J., and Kamin, H. (1982) Fed. Proc. 41, 888; and Batie, C.J., and Kamin, H. (1984) J. Biol. Chem. 259, 8832-8839). Premixing Fd with FNR was found to inhibit the reaction of the flavoprotein with NADP+ and with NADPH; thus, substrate binding may be ordered, NADP+ first, then Fd. FNRred and NADP+ very rapidly formed an FNRred X NADP+ complex with flavin to nicotinamide charge transfer bands. The Fdred X NADP+ complex then relaxed to an equilibrium species; the spectrum indicated a predominance of FNRox X NADPH charge-transfer complex. However, charge-transfer species were not observed during turnover; thus, their participation in catalysis of electron transfer from Fd to NADP+ remains uncertain. The catalytic rate of Fd to NADP+ electron transfer, as well as the rates of electron transfer from Fd to FNR, and from FNR to NADP+ were decreased when the reactants were in D2O; diaphorase activity was unaffected by solvent. On the basis of the data presented, a scheme for the catalytic mechanism of catalysis by FNR is presented.     

Arginyl groups involved in the binding of Anabaena ferredoxin--NADP+ reductase to NADP+ and to ferredoxin

Chemical modification of ferredoxin--NADP+ reductase from the cyanobacteria Anabaena has been performed using the alpha-dicarbonyl reagent phenylglyoxal. Inactivation of both the diaphorase and cytochrome-c reductase activities, characteristic of the enzyme, indicates the involvement of one or more arginyl residues in the catalytic process of the enzyme. The determination of the rate constants for the inactivation process under different conditions, including those in which substrates, NADP+ and ferredoxin, as well as other NADP+ analogs were present, indicates the involvement of two different groups in the inactivation process, one that reacts very rapidly with the reagent (kobs = 8.3 M-1 min-1) and is responsible for the binding of NADP+, and a second less reactive group (kobs = 0.9 M-1 min-1), that is involved in the binding of ferredoxin. Radioactive labeling of the enzyme with [14C]phenylglyoxal confirms that two groups are modified while amino acid analysis of the modified protein indicates that the modified groups are arginine residues. The identification of the amino acid residues involved in binding and catalysis of the substrates of ferredoxin--NADP+ reductase will help to elucidate the mechanism of the reaction catalyzed by this important enzyme.     

Interactions between spinach ferredoxin and other electron carriers. The involvement of a ferredoxin:cytochrome c complex in the ferredoxin-linked cytochrome c reductase activity of ferredoxin:NADP+ oxidoreductase.

The trinitrophenylation of a single amino group of spinach ferredoxin abolishes its ability to inhibit the diaphorase activity of the flavoprotein, ferredoxin:NADP oxidoreductase (EC 1.6.7.1); in contrast, the ability of ferredoxin to participate in the ferredoxin-linked cytochrome c reductase activity catalyzed by the flavoprotein is unaffected. Comparison with previously published results [Davis, D. J., and San Pietro, A. (1977) Biochem. Biophys. Res. Commun.74, 33–40]indicates that the site of interaction between ferredoxin and the flavoprotein resulting in inhibition if diaphorase activity is responsible for the spectrally observable 1:1 complex between the two proteins and is identical to the site of ferredoxin involvement in NADP photoreduction. The role of ferredoxin in the ferredoxin-linked cytochrome c reductase activity of the flavoprotein has been reexamined under conditions were the entire electron-accepting system (rather than just the ferredoxin component) is rate limiting. The data support a mechanism by which ferredoxin can bind either to the flavoprotein or to cytochrome c, and the ferredoxin:cytochrome c complex serves as the true substrate for reduction by the flavoprotein. Furthermore, Chromatographic evidence is presented for the formation of complexes between ferredoxin and cytochrome c.     

Electron acceptor specificity of ferredoxin (flavodoxin):NADP+ oxidoreductase from Escherichia coli

Reduced flavodoxin I (Fld1) is required in Escherichia coli for reductive radical generation in AdoMet-dependent radical enzymes and reductive activation of cobalamin-dependent methionine synthase. Ferredoxin (Fd) and flavodoxin II (Fld2) are also present, although their precise roles have not been ascertained. Ferredoxin (flavodoxin):NADP+ oxidoreductase (FNR) was discovered in E. coli as an NADPH-dependent reductant of Fld1 that facilitated generation of active methionine synthase in vitro; FNR and Fld1 will also supply electrons for the reductive cleavage of AdoMet essential for generating protein or substrate radicals in pyruvate formate-lyase, class III ribonucleotide reductase, biotin synthase, and, potentially, lipoyl synthase. As part of ongoing efforts to understand the various redox pathways that will support AdoMet-dependent radical enzymes in E. coli, we have examined the relative specificity of E. coli FNR for Fd, Fld1, and Fld2. While FNR will reduce all three proteins, Fd is the kinetically and thermodynamically preferred partner. Fd binds to FNR with high affinity (K(d)相似文献   

Thermal inactivation of reduced ferredoxin (flavodoxin):NADP+ oxidoreductase from Escherichia coli

Ferredoxin (flavodoxin):NADP+ oxidoreductase (FNR) is an essential enzyme that supplies electrons from NADPH to support flavodoxin-dependent enzyme radical generation and enzyme activation. FNR is a monomeric enzyme that contains a non-covalently bound FAD cofactor. We report that reduced FNR from Escherichia coli is subject to inactivation due to unfolding of the protein and dissociation of the FADH(2) cofactor at 37 degrees C. The inactivation rate is temperature-dependent in a manner that parallels the thermal unfolding of the protein and is slowed by binding of ferredoxin or flavodoxin. Understanding factors that minimize inactivation is critical for utilizing FNR as an accessory protein for S-adenosyl-L-methionine-dependent radical enzymes and manipulating FNR as an electron source for biotechnology applications.     

The haemoglobin-like protein (HMP) of Escherichia coli has ferrisiderophore reductase activity and its C-terminal domain shares homology with ferredoxin NADP+ reductases.

Three soluble ferrisiderophore reductases (FsrA, FsrB and FsrC) were detected in Escherichia coli. FsrB was purified and identified as the haemoglobin-like protein (HMP) by size and N-terminal sequence analyses. HMP was previously isolated as a dihydropteridine reductase and is now shown to have ferrisiderophore reductase activity. Database searches revealed that the C-terminal region of HMP (FsrB) is homologous to members of a family of flavoprotein oxidoreductases which includes ferredoxin NADP+ reductase (FNR). The combination of FNR-like and haemoglobin-like regions in HMP (FsrB) represents a novel pairing of functionally and structurally distinct domains. Structure-function properties of other FNR-like proteins, including LuxG and VanB, are also discussed.     

Binding of ferredoxin NADP+ oxidoreductase (FNR) to plant photosystem I

The binding of FNR to PSI has been postulated long ago, however, a clear evidence is still missing. In this work, using isothermal titration calorimetry (ITC), we found that FNR binds to photosystem I with its light harvesting complex I (PSI-LHCI) from C. reinhardtii with a 1:1 stoichiometry, a Kd of ~0.8 μM and ?H of ?20.7 kcal/mol. Titrations at different temperatures were used to determine the heat capacity change, ?CP, of the binding, through which the size of the interface area between the proteins was assessed as ~3000 Ų. In a different set of ITC experiments, introduction of various sucrose concentrations was used to estimate that ~95 water molecules are released to the solvent. These observations support the notion of a binding site shared by few of the photosystem I - light harvesting complex I (PSI-LHCI) subunits in addition to PsaE. Based on these results, a hypothetical model was built for the binding site of FNR at PSI, using known crystallographic structures of: cyanobacterial PSI in complex with ferredoxin (Fd), plant PSI-LHCI and Fd:FNR complex from cyanobacteria. FNR binding site location is proposed to be at the foot of the stromal ridge and above the inner LHCI belt. It is expected to form contacts with PsaE, PsaB, PsaF and at least one of the LHCI. In addition, a ~4.5-fold increased affinity between FNR and PSI-LHCI under crowded 1 M sucrose environment led us to conclude that in C. reinhardtii FNR also functions as a subunit of PSI-LHCI.     

Amino acid sequence of spinach ferredoxin:NADP+ oxidoreductase

The amino acid sequence of spinach ferredoxin: NADP+ oxidoreductase was determined by using overlapping sets of peptides derived by cleavage at arginyl or methionyl residues. The protein from different preparations varied in its length at the amino terminus. In the longest form the amino terminus is blocked with a pyroglutamyl residue, as determined by NMR. A single disulfide bond was placed between cysteine residues 132 and 137. The 314-residue sequence corresponds to a molecular weight of 35 317. The carboxyl-terminal half of the sequence has been fit to the electron density map of the NADP binding domain, revealing that this portion of the chain forms a typical nucleotide binding fold.     

Monoclonal antibody studies of ferredoxin:NADP+ oxidoreductase.

Eleven independent monoclonal antibodies, all IgG's, have been raised against the ferredoxin:NADP+ oxidoreductase of spinach leaves. All 11 monoclonal antibodies were able to produce substantial inhibition of the NADPH to 2,6-dichlorophenol indophenol (DCPIP) diaphorase activity of the enzyme, but none of the antibodies produced any significant inhibition of electron flow from NADPH to ferredoxin catalyzed by the enzyme. Spectral perturbation assays were used to demonstrate that antibody interaction with NADP+ reductase did not interfere significantly with the binding of either ferredoxin or NADP+ to the enzyme. Ultrafiltration binding assays were used to confirm that the monoclonal antibodies did not interfere with complex formation between ferredoxin and the enzyme. These results have been interpreted in terms of the likely presence of one or more highly antigenic epitopes at the site where the nonphysiological electron acceptor, DCPIP, binds to the enzyme. Furthermore, the results suggest that the site where DCPIP is reduced differs from both of the two separate sites at which the two physiological substrates, ferredoxin and NADP+/NADPH, are bound.     

Tryptophan fluorescence studies of ferredoxin:NADP reductase indicate the presence of tryptophan in or near the ferredoxin binding site

The tryptophan fluorescence properties of the flavoprotein ferredoxin:NADP reductase have been examined. Although not sensitive to changes in pH or salt concentration, the tryptophan fluorescence is affected by the presence of substrates for the flavoprotein. While NADP addition results in a slight quenching of the fluorescence, ferredoxin decreases the fluorescence by nearly 50%, suggesting the presence of tryptophan in or near the ferredoxin binding site. Titration of this effect gives a dissociation constant for the ferredoxin: flavoprotein complex which is similar to that obtained by spectral perturbations. This approach has also been used to demonstrate that a chemically modified ferredoxin which does not produce spectral perturbations when added to flavoprotein is capable of interacting with the flavoprotein although with a higher dissociation constant than for native ferredoxin.