Molecular interaction of ferredoxin and ferredoxin-NADP+ reductase from human malaria parasite

The malaria parasite possesses plant-type ferredoxin (Fd) and ferredoxin-NADP(+) reductase (FNR) in a plastid-derived organelle called the apicoplast. This Fd/FNR redox system, which potentially provides reducing power for essential biosynthetic pathways in the apicoplast, has been proposed as a target for the development of specific new anti-malarial agents. We studied the molecular interaction of Fd and FNR of human malaria parasite (Plasmodium falciparum), which were produced as recombinant proteins in Escherichia coli. NMR chemical shift perturbation analysis mapped the location of the possible FNR interaction sites on the surface of P. falciparum Fd. Site-specific mutation of acidic Fd residues in these regions and the resulting analyses of electron transfer activity and affinity chromatography of those mutants revealed that two acidic regions (a region including Asp26, Glu29 and Glu34, and the other including Asp65 and Glu66) dominantly contribute to the electrostatic interaction with P. falciparum FNR. The combination of Asp26/Glu29/Glu34 conferred a larger contribution than that of Asp65/Glu66, and among Asp26, Glu29 and Glu34, Glu29 was shown to be the most important residue for the interaction with P. falciparum FNR. These findings provide the basis for understanding molecular recognition between Fd and FNR of the malaria parasite.     

Anthracycline antibiotic reduction by spinach ferredoxin-NADP+ reductase and ferredoxin

Spinach NADPH:ferredoxin oxidoreductase (EC 1.6.7.1) catalyzes the NADPH-dependent reduction of the anthracyclines daunomycin, aclacinomycin A, and nogalamycin and their respective 7-deoxyanthracyclinones. Under anaerobic conditions, the endogenous rate of O2 reduction by NADPH catalyzed by ferredoxin reductase (0.12 s-1 at pH 7.4) is augmented by the anthracyclines and 7-deoxyanthracyclinones. The catalytic constants are approximately equivalent for this augmentation for all substrates (approximate V of 2 s-1 and KM of 75 microM). Both O2- and H2O2 are made. Under anaerobic conditions, anthracycline reduction catalyzed by ferredoxin reductase results in the elimination of the C-7 substituent to provide a quinone methide intermediate. Following tautomerization by C-7 protonation, 7-deoxyanthracyclinones are obtained. Under appropriate conditions these may be further reduced to the 7-deoxyanthracyclinone hydroquinones. For daunomycin, the quinone methide is formed rapidly after reduction and is easily monitored at 600 nm. It may react with electrophiles other than H+, as demonstrated by its competitive trapping by p-carboxybenzaldehyde. It may also react with nucleophiles, as demonstrated by its competitive trapping by N-acetylcysteine. For aclacinomycin, quinone methide formation is also rapid although no distinct transient near 600 nm occurs. In addition to protonation, it reacts with itself providing the 7,7'-dimer. With ethyl xanthate as a thiolate nucleophile, adducts derived from addition to C-7 are obtained. For nogalamycin, quinone methide formation is not rapid. Nogalamycin is reduced to its hydroquinone, which slowly converts in a first-order process [k = (1.2 +/- 0.2) X 10(-3) s-1, pH 8.0, 30 degrees C] to the quinone methide, which is then quenched by protonation. Spinach ferredoxin in its reduced form is chemically competent for anthracycline reduction. Its effect on both the aerobic and anaerobic reactions catalyzed by ferredoxin reductase is to increase severalfold the overall velocity for anthracycline reduction. In conclusion, the aerobic reaction pathways for the anthracyclines as mediated by ferredoxin reductase are remarkably similar, while the anaerobic reactions are remarkably different. If these anthracyclines exert their antitumor activity by a common anaerobic pathway, it is most likely that the pathway is determined by the properties of the anthracycline as complexed to its in vivo target. The behavior of ferredoxin further suggests that not only low-potential flavin centers but also iron-sulfur centers should be regarded as important loci for anthracycline reductive activation.     

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.     

Novel forms of ferredoxin and ferredoxin-NADP reductase from spinach roots

Ferredoxin and the enzyme catalyzing its reduction by NADPH, ferredoxin-NADP reductase (ferredoxin-NADP+ oxidoreductase or FNR), were found to be present in roots of spinach (Spinacia oleracea). Localization experiments with endosperm of germinating castor beans (Ricinus communis), a classical nonphotosynthetic tissue for cell fractionation studies, confirmed that ferredoxin and FNR are localized in the plastid fraction. Both proteins were purified from spinach roots and found to resemble their leaf counterparts in activity, spectral properties, and complex formation, but to differ in amino acid composition and amino terminal sequence. The results indicate that the primary structures of the FNR and ferredoxin of spinach roots differ from that of the corresponding leaf proteins. Together with earlier findings, the present results provide evidence that nonphotosynthetic plastids, including those of roots, are capable of reducing ferredoxin with heterotrophically generated NADPH.     

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.     

Complex formation between ferredoxin and ferredoxin-NADP+ reductase from Anabaena PCC 7119: cross-linking studies.

Ferredoxin-NADP+ reductase and ferredoxin from the cyanobacterium Anabaena PCC 7119 have been covalently cross-linked by incubation with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide. The covalent adduct, which shows a molecular mass consistent with a 1:1 stoichiometry of the two proteins, maintains nearly 60% of the NADPH-cytochrome c reductase activity of the enzyme saturated with ferredoxin and this value is considerably higher than when equimolar amounts of both proteins are assayed. No ternary complexes with Anabaena flavodoxin or horse heart cytochrome c were formed, suggesting that the binding site on the enzyme is the same for ferredoxin and flavodoxin and that ferredoxin-NADP+ reductase and cytochrome c bind at a common site on ferredoxin. In the noncovalent complex, titrated at pH 7, the oxidation-reduction potential of ferredoxin becomes 15 mV more negative and that of ferredoxin-NADP+ reductase 27 mV more positive compared to the proteins alone. When covalently linked, the midpoint potential of the enzyme has a value similar to that in the noncovalent complex, while the ferredoxin potential is 20 mV more positive compared to ferredoxin alone. The changes in redox potentials have been used to estimate the dissociation constants for the interaction of the different redox forms of the proteins, based on the value of 1.21 microM calculated for the oxidized noncovalent complex.     

Reactions of antibodies against ferredoxin, ferredoxin-NADP+ reductase and plastocyanin with spinach chloroplasts

Purified antisera against ferredoxin, ferredoxin-NADP+ reductase and plastocyanin agglutinated osmotically shocked and washed spinach chloroplasts, prepared according to standard procedures. The monomeric antibody (immunoglobulin G fraction) of the reductase antiserum agglutinated chloroplasts specifically and directly, indicating that protruding structures (for example, the coupling factor) do not act as steric hindrances as has been suggested. With ferredoxin antiserum, the presence of a pentameric antibody (immunoglobulin M fraction) was obligatory to observe a positive agglutination reaction. Immunoglobulin G only inhibited ferredoxin-dependent reactions, like NADP+-photoreduction, but did not cause agglutination. Ferredoxin seems to be located in depressions of the membrane, possibly caused by a partial release of this protein in shocked chloroplasts. Similar results were obtained with purified immunoglobulins from a plastocyanin antiserum. Again the immunoglobulin G fraction inhibited electron transport reactions catalyzed by plastocyanin, whereas immunoglobulin M showed a positive agglutination, but had no influence on electron transport. It is concluded that ferredoxin, ferredoxin-NADP+ reductase and plastocyanin are peripheral electron transport components, located at the outer thylakoid membrane.     

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.     

Apicomplexan parasites possess distinct nuclear-encoded, but apicoplast-localized, plant-type ferredoxin-NADP+ reductase and ferredoxin

In searching for nuclear-encoded, apicoplast-localized proteins we have cloned ferredoxin-NADP(+) reductase from Toxoplasma gondii and a [2Fe-2S] ferredoxin from Plasmodium falciparum. This chloroplast-localized redox system has been extensively studied in photosynthetic organisms and is responsible for the electron transfer from photosystem I to NADP+. Besides this light-dependent reaction in nonphotosynthetic plastids (e.g. from roots), electrons can also flow in the reverse direction, from NADPH to ferredoxin, which then serves as an important reductant for various plastid-localized enzymes. These plastids possess related, but distinct, ferredoxin-NADP+ reductase and ferredoxin isoforms for this purpose. We provide phylogenetic evidence that the T. gondii reductase is similar to such nonphotosynthetic isoforms. Both the P. falciparum [2Fe-2S] ferredoxin and the T. gondii ferredoxin-NADP+ reductase possess an N-terminal bipartite transit peptide domain typical for apicoplast-localized proteins. The recombinant proteins were obtained in active form, and antibodies raised against the reductase recognized two bands on Western blots of T. gondii tachyzoite lysates, indicative of the unprocessed and native form, respectively. We propose that the role of this redox system is to provide reduced ferredoxin, which might then be used for fatty acid desaturation or other biosynthetic processes yet to be defined. Thus, the interaction of these two proteins offers an attractive target for drug intervention.     

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.     

A ferredoxin Arg-Glu pair important for efficient electron transfer between ferredoxin and ferredoxin-NADP(+) reductase

In order to elucidate the importance of a ferredoxin (Fd) Arg-Glu pair involved in dynamic exchange from intra- to intermolecular salt bridges upon complex formation with ferredoxin-NADP(+) oxidoreductase (FNR), Equisetum arvense FdI and FdII were investigated as normal and the pair-lacking Fd, respectively. The FdI mutant lacking this pair was unstable and rapidly lost the [2Fe-2S] cluster. The catalytic constant (k(cat)) of the electron transfer for FdI is 5.5 times that for FdII and the introduction of this pair into FdII resulted in the increase of k(cat) to a level comparable to that for FdI, demonstrating directly that the Arg-Glu pair is important for efficient electron transfer between Fd and FNR.     

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.     

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.     

Structure of the electron transfer complex between ferredoxin and ferredoxin-NADP(+) reductase

All oxygenic photosynthetically derived reducing equivalents are utilized by combinations of a single multifuctional electron carrier protein, ferredoxin (Fd), and several Fd-dependent oxidoreductases. We report the first crystal structure of the complex between maize leaf Fd and Fd-NADP(+) oxidoreductase (FNR). The redox centers in the complex--the 2Fe-2S cluster of Fd and flavin adenine dinucleotide (FAD) of FNR--are in close proximity; the shortest distance is 6.0 A. The intermolecular interactions in the complex are mainly electrostatic, occurring through salt bridges, and the interface near the prosthetic groups is hydrophobic. NMR experiments on the complex in solution confirmed the FNR recognition sites on Fd that are identified in the crystal structure. Interestingly, the structures of Fd and FNR in the complex and in the free state differ in several ways. For example, in the active site of FNR, Fd binding induces the formation of a new hydrogen bond between side chains of Glu 312 and Ser 96 of FNR. We propose that this type of molecular communication not only determines the optimal orientation of the two proteins for electron transfer, but also contributes to the modulation of the enzymatic properties of FNR.     

Electron transfer of site-specifically cross-linked complexes between ferredoxin and ferredoxin-NADP(+) reductase

Ferredoxin (Fd) and Fd-NADP(+) reductase (FNR) are redox partners responsible for the conversion between NADP(+) and NADPH in the plastids of photosynthetic organisms. Introduction of specific disulfide bonds between Fd and FNR by engineering cysteines into the two proteins resulted in 13 different Fd-FNR cross-linked complexes displaying a broad range of activity to catalyze the NADPH-dependent cytochrome c reduction. This variability in activity was thought to be mainly due to different levels of intramolecular electron transfer activity between the FNR and Fd domains. Stopped-flow analysis revealed such differences in the rate of electron transfer from the FNR to Fd domains in some of the cross-linked complexes. A group of the cross-linked complexes with high cytochrome c reduction activity comparable to dissociable wild-type Fd/FNR was shown to assume a similar Fd-FNR interaction mode as in the native Fd:FNR complex by analyses of NMR chemical shift perturbation and absorption spectroscopy. However, the intermolecular electron transfer of these cross-linked complexes with two Fd-binding proteins, nitrite reductase and photosystem I, was largely inhibited, most probably due to steric hindrance by the FNR moiety linked near the redox center of the Fd domain. In contrast, another group of the cross-linked complexes with low cytochrome c reduction activity tends to mediate higher intermolecular electron transfer activity. Therefore, reciprocal relationship of intramolecular and intermolecular electron transfer abilities was conferred by the linkage of Fd and FNR, which may explain the physiological significance of the separate forms of Fd and FNR in chloroplasts.     

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.