Giuliana Zanetti

A productive NADP+ binding mode of ferredoxin-NADP+ reductase revealed by protein engineering and crystallographic studies.

The flavoenzyme ferredoxin–NADP+ reductase (FNR) catalyzes the production of NADPH during photosynthesis. Whereas the structures of FNRs from spinach leaf and a cyanobacterium as well as many of their homologs have been solved, none of these studies has yielded a productive geometry of the flavin–nicotinamide interaction. Here, we show that this failure occurs because nicotinamide binding to wild type FNR involves the energetically unfavorable displacement of the C-terminal Tyr side chain. We used mutants of this residue (Tyr 308) of pea FNR to obtain the structures of productive NADP+ and NADPH complexes. These structures reveal a unique NADP+ binding mode in which the nicotinamide ring is not parallel to the flavin isoalloxazine ring, but lies against it at an angle of ~30°, with the C4 atom 3 Å from the flavin N5 atom.

Structural and functional diversity of ferredoxin-NADP(+) reductases

Although all ferredoxin-NADP(+) reductases (FNRs) catalyze the same reaction, i.e. the transfer of reducing equivalents between NADP(H) and ferredoxin, they belong to two unrelated families of proteins: the plant-type and the glutathione reductase-type of FNRs. Aim of this review is to provide a general classification scheme for these enzymes, to be used as a framework for the comparison of their properties. Furthermore, we report on some recent findings, which significantly increased the understanding of the structure-function relationships of FNRs, i.e. the ability of adrenodoxin reductase and its homologs to catalyze the oxidation of NADP(+) to its 4-oxo derivative, and the properties of plant-type FNRs from non-photosynthetic organisms. Plant-type FNRs from bacteria and Apicomplexan parasites provide examples of novel ways of FAD- and NADP(H)-binding. The recent characterization of an FNR from Plasmodium falciparum brings these enzymes into the field of drug design.

Reconstitution of an apicoplast-localised electron transfer pathway involved in the isoprenoid biosynthesis of Plasmodium falciparum.

In the malaria parasite Plasmodium falciparum isoprenoid precursors are synthesised inside a plastid‐like organelle (apicoplast) by the mevalonate independent 1‐deoxy‐d‐xylulose‐5‐phosphate (DOXP) pathway. The last reaction step of the DOXP pathway is catalysed by the LytB enzyme which contains a [4Fe–4S] cluster. In this study, LytB of P. falciparum was shown to be catalytically active in the presence of an NADPH dependent electron transfer system comprising ferredoxin and ferredoxin‐NADP+ reductase. LytB and ferredoxin were found to form a stable protein complex. These data suggest that the ferredoxin/ferredoxin‐NADP+ reductase redox system serves as the physiological electron donor for LytB in the apicoplast of P. falciparum.

Molecular heterogeneity of ferredoxin-NADP+ reductase from spinach leaves

Ferredoxin-NADP+ reductase (NADPH: ferredoxin oxidoreductase, EC 1.6.7.1) from spinach leaves has been purified according to a new procedure. The enzyme shows the presence of five molecular forms as identified by isoelectric focusing, namely a, b, c, d and e with pI values of 6.0, 5.5, 5.2, 5.0 and 4.8, respectively. All the bands are catalytically active and are clearly identifiable after the first steps of the purification procedure. The basic pattern of the ferredoxin-NADP+ reductase forms is the same whether extracted from one or many spinach plants and is not affected by the different purification procedures used. Two distinct classes of molecular weight have been found for the isolated forms b, c and d as measured by sodium dodecyl sulphate electrophoresis, with values of 33 000-34 000 for the first and 36 000-38 000 for the later two forms. Gel electrophoresis in non-denaturing media at different gel concentrations gives the same order of molecular weight values, thus ruling out the possibility that the native enzyme is a dimer, as has been reported by Schneeman, R. and Krogmann, D.W. ((1975) J. Biol. Chem. 250, 4965-4971). No significant kinetic differences were detectable for the isolated forms of ferredoxin-NADP+ reductase.

[22] Ferredoxin-NADP+ oxidoreductase

Publisher Summary This chapter focuses on enzyme ferredoxin–NADP + oxidoreductase. The flavoprotein ferredoxin–NADP + reductase (EC 1.6.7.1) is a membrane-bound component of the photosynthetic electron-transport system. Extensive purification, crystallization, and kinetic and structural studies have been carried out on the spinach enzyme. Several catalytic roles have been shown for this enzyme. They include (a) the photoreduction of NADP + by ferredoxin as electron donor, (b) the reduction of NAD + by NADPH or its analogs (transhydrogenase), (c) the oxidation of NADPH by K 3 Fe(CN) 6 or dichlorophenolindophenol (DCPIP) or 2-( p -iodophenyl)-3-nitrophenyl-5-phenyltetrazolium chloride (INT) (diaphorase), and (d) the reduction of cytochrome f by NADPH. The more reliable assay methods involve the reduction of ferredoxin or K 3 Fe(CN) 6 by NADPH in the presence of a regenerating system; alternative procedures have been described according to the different catalytic roles of the enzyme. A method is described for purification of ferredoxin–NADP + reductase that has the advantage of shortening the working time and avoiding the use of large volumes of acetone, without decreasing the yield and the specific activity. An overview of the properties of the enzyme is also detailed in the chapter.

Structure of the mutant E92K of [2Fe-2S] ferredoxin I from Spinacia oleracea at 1.7 A resolution.

Ferredoxin I (Fd I) from Spinacia oleracea is composed of 97 amino-acid residues and a [2Fe-2S] cluster. The crystal structure of the E92K mutant of Fd I was solved by molecular replacement and refined to an R factor of 19.6% for 11755 reflections at 1.7 A resolution. The overall structure and the active centre of spinach Fd is highly conserved with respect to ferredoxins of known structure. The E92K mutation appears to disturb a hydrogen-bond network which stabilizes the loop bearing the [2Fe-2S] cluster. This observation provides a rationale for the reduced electron-transfer efficiency displayed by the E92K mutant. Inspection of the crystal packing reveals that the side chain of Lys92 is engaged in an intermolecular interaction with Asp26 of a symmetry-related molecule. This feature may explain why only the mutant E92K and not wild-type Fd I could be successfully crystallized.

Rabbit liver glutathione reductase. Purification and properties

Abstract Hepatic glutathione reductase can be obtained in relatively good amounts from rabbit by a procedure which is fairly simple and sufficiently rapid. The purified flavoprotein shows absorbance ratios at 274, 379, and 463 nm of 8.2:0.92:1.0, respectively; the FAD fluorescence is nearly completely quenched by the protein. Gradient ultracentrifugation and sodium dodecyl sulfate gel electrophoresis indicate that the enzyme is a dimer, consisting of subunits of about 56,000 molecular weight; flavin content suggests one FAD per chain. Gel filtration under a variety of conditions, on the other hand, yields a molecular weight in the range 56,000–67,000. It is proposed that rabbit liver glutathione reductase can be active also as monomer. Kinetic parameters of the enzyme have been determined under optimal conditions. The rabbit liver glutathione reductase is, at physiological pH, absolutely specific for NADPH.

Involvement of lysine‐88 of spinach ferredoxin‐NADP+ reductase in the interaction with ferredoxin

A mutant of spinach ferredoxin‐NADP+ reductase, in which Lys‐88 has been changed to glutamine, has been obtained by site‐directed mutagenesis. The mutant enzyme was fully active as a diaphorase, but partially impaired in ferredoxin‐dependent cytochrome c reductase activity. By steady‐state kinetics, the K m for ferredoxin of the K88Q enzyme was found to have increased 10‐fold, whereas the k cat was unaffected by the amino acid replacement. The interaction between oxidized ferredoxin and the enzyme forms was also studied by spectrofluorimetric titration:K d values of 110 and 10 nM were determined for the mutant and wild‐type proteins, respectively. These data point out the importance of a positive charge at position 88 of the reductase for the interaction with ferredoxin, confirming previous cross‐linking studies.

The Plant-Type Ferredoxin-NADP + Reductase/Ferredoxin Redox System as a Possible Drug Target Against Apicomplexan Human Parasites

Apicomplexa are unicellular, obligate intracellular parasites of great medical importance. They include human pathogens like Plasmodium spp., the causative agent of malaria, and Toxoplasma gondii, an opportunistic parasite of immunosuppressed individuals and a common cause of congenital disease (toxoplasmosis). They alone affect several hundred million people worldwide so that new drugs, especially for plasmodial infections, are urgently needed. This review will focus on a recently emerged, potential drug target, a plant-type redox system consisting of ferredoxin-NADP(+) reductase (FNR) and its redox partner, ferredoxin (Fd). Both reside in an unique organelle of these parasites, named apicoplast, which is of algal origin. The apicoplast has been shown to be required for pathogen survival. In addition to other pathways already identified in this compartment, the FNR/Fd redox system represents a promising drug target because homologous proteins are not present in host organisms. Furthermore, a wealth of structural information exists on the closely related plant proteins, which can be exploited for structure-function studies of the apicomplexan protein pair. T. gondii and P. falciparum FNRs have been cloned, and the T. gondii enzyme was shown to be a flavoprotein active as a NADPH-dependent oxidoreductase. Both phylogenetic and biochemical analyses indicate that T. gondii FNR is similar in function to the isoform present in non-photosynthetic plastids whereby electron flow is from NADPH to oxidized Fd. The resulting reduced Fd is then presumably used as a reductant for various target enzymes whose nature is just starting to emerge. Among the likely candidates is the iron-sulfur cluster biosynthesis pathway, which is also located in the apicoplast and dependent on reducing power. Furthermore, lipoic acid synthase and enzymes of the isoprenoid biosynthetic pathway may be other conceivable targets. Since all these metabolic steps are vital for the parasite, blocking electron flow from FNR to Fd by inhibition of either FNR activity or its molecular interaction with Fd should also interfere with these pathways, ultimately killing the parasite. Although the three-dimensional structure of FNR from T. gondii is not yet known, experimental and computational evidence shows that apicomplexan and plant enzymes are very similar in structure. Furthermore, single amino acid changes can have profound effects on the enzyme activity and affinity for Fd. This knowledge may be exploited for the design of inhibitors of protein-protein interaction. On the other hand, specifically tailored NAD(P) analogues or mimetics based on previously described substances might be useful lead compounds for apicomplexan FNR inhibitors.

Purification and properties of cytochrome f from parsley leaves

Abstract A method for the extraction and purification of cytochrome f from parsley leaves is described. Cytochrome f is obtained in a high state of purity. A molecular weight of 245000 is indicated by Sephadex G-200 chromatography. The absorption spectrum of pure cytochrome f in the reduced state shows peaks at 554·5 mμ, 532.8 mμ, 524 mμ, 422 mμ and 330 mμ and a shoulder at 402 mμ. Cytochrome f possesses catalase (EC 1.11.1.6) activity, with a Kat. f value of 4.7 · 104.