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.     

Chemical modification and cross-linking as probes of regions on ferredoxin involved in its interaction with ferredoxin: NADP reductase

Ferredoxin which had been modified with glycine ethylester in the presence of a water-soluble carbodiimide to the extent of one carboxyl-group modified per ferredoxin was subjected to peptide mapping in an attempt to locate the site(s) of modification. The peptide mapping was done by HPLC and analysis of the resulting chromatogram allowed assignment of peaks to various segments in the amino acid sequences of the two isozymes of ferredoxin. The modified ferredoxin appeared to be a mixture of ferredoxin derivatives in which modification had occurred in three areas of the molecule. Although unable to identify the specific residues modified, it has been shown that modification is localized in the regions of residues 26-30, 65-70, and 92-94. The possibility that these regions of ferredoxin may define its binding site for ferredoxin: NADP reductase is discussed. Peptide mapping studies on a covalently linked adduct between ferredoxin and ferredoxin: NADP reductase also support these regions of ferredoxin as being important in the interaction between the two proteins.     

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.     

Ferredoxin:NADP+ oxidoreductase. Equilibria in binary and ternary complexes with NADP+ and ferredoxin

Ferredoxin:NADP+ oxidoreductase (ferredoxin: NADP+ reductase, EC 1.18.1.2) was shown to form a ternary complex with its substrates ferredoxin (Fd) and NADP(H), but the ternary complex was less stable than the separate binary complexes. Kd for oxidized binary Fd-ferredoxin NADP+ reductase complex was less than 50 nM; Kd(Fd) increased with NADP+ concentration, approaching 0.5-0.6 microM when the flavoprotein was saturated with NADP+ K(NADP+) also increased from about 14 microM to about 310 microM, on addition of excess Fd. The changes in Kd were consistent with negative cooperativity between the associations of Fd and NADP+ and with our unpublished observations which suggest that product dissociation is rate-limiting in the reaction mechanism. Similar interference in binding was observed in more reduced states; NADPH released much ferredoxin:NADP+ reductase from Fd-Sepharose whether the proteins were initially oxidized or reduced. Complexation between Fd and ferredoxin: NADP+ reductase was found to shield each center from paramagnetic probes; charge specificity suggested that the active sites of Fd and ferredoxin:NADP+ reductase were, respectively, negatively and positively charged.     

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.     

Inhibition of ferredoxin: NADP+ reductase activity by the hexacyanochromate (III) ion

The small inorganic complex Cr(CN)6(3-) is a clean inhibitor of the ferredoxin: NADP+ reductase-catalysed oxidation of reduced spinach ferredoxin by NADP+. Independent spectrophotometric measurements show that millimolar additions of Cr(CN)6(3-) to mixtures of ferredoxin and ferredoxin NADP+ reductase give a marked attenuation of the difference spectrum characteristic of ferredoxin-ferredoxin: NADP+ reductase complex formation. Since there is no evidence, from NMR studies, for significant binding of Cr(CN)6(3-) to ferredoxin, these results indicate that Cr(CN)6(3-) binds to ferredoxin: NADP+ reductase at a site which is crucial to its interaction with the electron-transfer protein. The effective kinetic binding constant for Cr(CN)6(3-), measured at low ferredoxin concentration, is 445 M-1 (ie Kdiss congruent to 2 mM) at 25 degrees, pH7.5, I = 0.10 M. With assumption of a simple electrostatic interaction, an enzyme domain with an effective charge of 3+/4+ is proposed.     

Effect of pH, salt, and coupling state on the interaction of ferredoxin with the chloroplast membrane

To determine if the interaction between ferredoxin and ferredoxin:NADP reductase is similar to the interaction between the purified proteins when the ferrodoxin:NADP reductase is membrane bound, the effect of pH, salt, and coupling state on the Km for ferredoxin in NADP reduction by chloroplast membranes has been examined. Increasing pH and salt concentrations as well as uncouplers all resulted in increases in the Km for ferredoxin. The pH and salt effects on the Km are similar to effects observed by others (C. Batie and H. Kamin (1981) J. Biol. Chem. 256, 7756-7763) on the dissociation constant for a complex between the two purified proteins, although the salt effect on the Km appears to be affected by the surface potential of the chloroplast membrane. These results suggest that the interaction between ferredoxin and the membrane-bound ferredoxin:NADP reductase is not greatly different from the interaction which has been characterized between the two purified proteins.     

Chemical modification of spinach ferredoxin: evidence for the involvement of a complex between ferredoxin and ferredoxin:NADP oxidoreductase in NADP photoreduction.

Spinach ferredoxin was modified chemically with trinitrobenzene sulfonic acid (TNBS), a reagent which reacts specifically with amino groups. The trinitrophenylated ferredoxin (TNP-Fd) can accept electrons from Photosystem I as indicated by its full activity in the photoreduction of cytochrome $\text{c}$. The modified protein is inactive, however, in the photoreduction of NADP and cannot form a complex with the flavoprotein, ferredoxin: NADP oxidoreductase. The data presented indicate that the inactivity of the modified protein is the result of modification of a single amino group.     

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     

On the specificity of pig adrenal ferredoxin (adrenodoxin) and spinach ferredoxin in electron-transfer reactions

Spinach leaf ferredoxin and ferredoxin:NADP oxidoreductase as well as pig adrenodoxin and adrenodoxin reductase have been purified to homogeneity. Ferredoxin-NADP reductase and adrenodoxin-NADP reductase can perform the same diaphorase reactions (dichloroindophenol, ferricyanide and cytochrome c reduction) albeit not with the same efficiency. Despite the differences in their redox potentials, animal and plant ferredoxins can be used as heterologous substrates by the ferredoxin-NADP reductases from both sources. In heterologous systems, however, the ferredoxin/adrenodoxin concentrations must be increased approximately 100-fold in order to reach rates similar to those obtained in homologous systems. Ferredoxin and adrenodoxin can form complexes with the heterologous reductases as demonstrated by binding experiments on ferredoxin-Sepharose or ferredoxin-NADP-reductase-Sepharose and by the realization of difference spectra. Adrenodoxin also weakly substitutes for ferredoxin in NADP photoreduction, and can be used as an electron carrier in the light activation of the chloroplastic enzyme NADP-dependent malate dehydrogenase. In addition adrenodoxin is a good catalyst of pseudocyclic photophosphorylation, but not of cyclic phosphorylation and can serve as a substrate of glutamate synthase. These results are discussed with respect to the known structures of plant and animals ferredoxins and their respective reductases.     

Interaction of ferredoxin and ferredoxin-NADP reductase with thylakoids

Ferredoxin-NADP reductase accounts for about 50% of the NADPH diaphorase activity of spinach leaf homogenates. The enzyme is bound to thylakoid membranes, but can be slowly extracted by aqueous buffers. Ferredoxin-NADP reductase can be extracted from the membranes by a 1- to 2-min treatment with a low concentration of trypsin. This treatment completely inactivates NADP photoreduction but does not affect electron transport from water to ferredoxin. It is shown that the inactivation is due to solubilization of ferredoxin-NADP reductase: the activity can be restored by addition of a very large excess of soluble enzyme in pure form. When ferredoxin-NADP reductase is added as a soluble enzyme after extraction or inactivation (by a specific antibody) of the membrane-bound enzyme, NADP photoreduction requires a very large excess of this enzyme, and the apparent K_m for ferredoxin is also increased. These observations are discussed as related to the interactions of thylakoids with ferredoxin-NADP reductase.     

Protein modulase appears to be a complex of ferredoxin, ferredoxin/thioredoxin reductase, and thioredoxin

Protein modulase and ferredoxin/thioredoxin reductase are soluble proteins that have been suggested to catalyze the light-dependent modulation of enzyme activity in the stromal compartment of the chloroplast. Protein modulase is active in vitro without additional ferredoxin and thioredoxin, whereas ferredoxin/thioredoxin reductase requires additional ferredoxin and thioredoxin. We hypothesize that protein modulase is a complex protein composed of ferredoxin/thioredoxin reductase, ferredoxin, and thioredoxin. In reconstituted chloroplast systems, antiserum directed against ferredoxin, at concentrations sufficient to inhibit the photoreduction of NADP, had no effect on light modulation. Antiserum directed against thioredoxin gave variable results: one batch of polyclonal antibodies inhibited light modulation, another was stimulatory, and another was without effect. These results suggest that the ferredoxin and thioredoxin active in light modulation are not free in solution. Furthermore, molecular sieve chromatography of stromal proteins results in the elution of four species that catalyze light modulation. Based on whether or not ferredoxin and/or thioredoxin must be added for activity, these four species have been tentatively identified as protein modulase, a complex of ferredoxin/thioredoxin reductase and ferredoxin, a complex of ferredoxin/thioredoxin reductase and thioredoxin, and ferredoxin/thioredoxin reductase. That is, the four correspond to all the possible combinations of ferredoxin, ferredoxin/thioredoxin reductase, and thioredoxin. We suggest that buffer ionic strength affects the interactions among these proteins and in part determines the fate of the protein modulase complex in vitro.     

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.     

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.     

Ferredoxin-NADP+ reductase. Kinetics of electron transfer, transient intermediates, and catalytic activities studied by flash-absorption spectroscopy with isolated photosystem I and ferredoxin

The electron transfer cascade from photosystem I to NADP+ was studied at physiological pH by flash-absorption spectroscopy in a Synechocystis PCC6803 reconstituted system comprised of purified photosystem I, ferredoxin, and ferredoxin-NADP+ reductase. Experiments were conducted with a 34-kDa ferredoxin-NADP+ reductase homologous to the chloroplast enzyme and a 38-kDa N-terminal extended form. Small differences in kinetic and catalytic properties were found for these two forms, although the largest one has a 3-fold decreased affinity for ferredoxin. The dissociation rate of reduced ferredoxin from photosystem I (800 s(-1)) and the redox potential of the first reduction of ferredoxin-NADP+ reductase (-380 mV) were determined. In the absence of NADP+, differential absorption spectra support the existence of a high affinity complex between oxidized ferredoxin and semireduced ferredoxin-NADP+ reductase. An effective rate of 140-170 s(-1) was also measured for the second reduction of ferredoxin-NADP+ reductase, this process having a rate constant similar to that of the first reduction. In the presence of NADP+, the second-order rate constant for the first reduction of ferredoxin-NADP+ reductase was 20% slower than in its absence, in line with the existence of ternary complexes (ferredoxin-NADP+ reductase)-NADP+-ferredoxin. A single catalytic turnover was monitored, with 50% NADP+ being reduced in 8-10 ms using 1.6 microM photosystem I. In conditions of multiple turnover, we determined initial rates of 360-410 electrons per s and per ferredox-in-NADP+ reductase for the reoxidation of 3.5 microM photoreduced ferredoxin. Identical rates were found with photosystem I lacking the PsaE subunit and wild type photosystem I. This suggests that, in contrast with previous proposals, the PsaE subunit is not involved in NADP+ photoreduction.     

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.     

Porton uptake by isolated chloroplasts during cyclic and non-cyclic electron transport catalyzed by photosystem I in the presence of ferredoxin]

Proton uptake by isolated chloroplasts during cyclic electron transport in the presence of ferredoxin and under NADP+ reduction from the ascorbate--TMPD donor pair under anaerobic conditions was studied. It was found that during cyclic transport the proton uptake is less intensive than under NADP+ reduction. In the presence of ATP the proton uptake is increased in the first case and is decreased in the second one. During cyclic transport in the presence of gramicidin D the proton uptake is completely suppressed and under NADP+ reduction is decreased down to 0,08--0,09 mk equiv H+ per mg of chlorophyll, irrespective of ferredoxin or NADP+ concentrations. The role of ferredoxin NADP+ reductase in a proton uptake by thylakoids is discussed.     

NMR study of the electron transfer complex of plant ferredoxin and sulfite reductase: mapping the interaction sites of ferredoxin

Plant ferredoxin serves as the physiological electron donor for sulfite reductase, which catalyzes the reduction of sulfite to sulfide. Ferredoxin and sulfite reductase form an electrostatically stabilized 1:1 complex for the intermolecular electron transfer. The protein-protein interaction between these proteins from maize leaves was analyzed by nuclear magnetic resonance spectroscopy. Chemical shift perturbation and cross-saturation experiments successfully mapped the location of two major interaction sites of ferredoxin: region 1 including Glu-29, Glu-30, and Asp-34 and region 2 including Glu-92, Glu-93, and Glu-94. The importance of these two acidic patches for interaction with sulfite reductase was confirmed by site-specific mutation of acidic ferredoxin residues in regions 1 and 2, separately and in combination, by which the ability of mutant ferredoxins to transfer electrons and bind to sulfite reductase was additively lowered. Taken together, this study gives a clear illustration of the molecular interaction between ferredoxin and sulfite reductase. We also present data showing that this interaction surface of ferredoxin significantly differs from that when ferredoxin-NADP(+) reductase is the interaction partner.     

Affinity chromatography of Anacystis nidulans ferredoxin nitrate reductase and NADP reductase on reduced ferredoxin-Sepharose.

The behavior of two ferredoxin-dependent enzymes—nitrate reductase and NADP reductase—fromAnacystis nidulans on a ferredoxin-Sepharose gel was examined. The oxidized gel-bound ferredoxin exhibited very low affinity for these enzymes but effectively bound both nitrate reductase and NADP reductase when reduced by dithionite. Selective procedures are described for the clution of each of these two enzymes from the reduced ferredoxin-Sepharose gel. These simple methods allow substantial purification of both enzymes.     

Thermal properties of NADP:ferredoxin oxidoreductase and ferredoxin isolated from a thermophilic blue-green alga

NADP:ferredoxin oxidoreductase (EC. 1.6.7.1.) isolated from a thermophilic blue-green alga, Synechococcus sp., was stable at temperatures up to 65°C. The diaphorase and cytochrome c reductase activities of the enzyme were low at 25°C but increased with elevated temperature to reach a maximum at about 60°C. The pH-profile of the diaphorase activity showed a peak at pH 9.0 at 55°C, whereas the activity was largely independent of pH at 25°C. High concentrations of NaCl suppressed activity at both high and low temperatures. In the cytochrome c reductase activity catalyzed by the enzyme, ferredoxin served as an electron carrier in a temperature-insensitive manner over a wide range of temperature. The results support the view that the optimum and the upper limiting temperatures for photosynthesis in this alga are related to thermal properties of proteins.