Two functionally distinct NADP+-dependent ferredoxin oxidoreductases maintain the primary redox balance of Pyrococcus furiosus.

Electron bifurcation has recently gained acceptance as the third mechanism of energy conservation in which energy is conserved through the coupling of exergonic and endergonic reactions. A structure-based mechanism of bifurcation has been elucidated recently for the flavin-based enzyme NADH-dependent ferredoxin NADP+ oxidoreductase I (NfnI) from the hyperthermophillic archaeon Pyrococcus furiosus. NfnI is thought to be involved in maintaining the cellular redox balance, producing NADPH for biosynthesis by recycling the two other primary redox carriers, NADH and ferredoxin. The P. furiosus genome encodes an NfnI paralog termed NfnII, and the two are differentially expressed, depending on the growth conditions. In this study, we show that deletion of the genes encoding either NfnI or NfnII affects the cellular concentrations of NAD(P)H and particularly NADPH. This results in a moderate to severe growth phenotype in deletion mutants, demonstrating a key role for each enzyme in maintaining redox homeostasis. Despite their similarity in primary sequence and cofactor content, crystallographic, kinetic, and mass spectrometry analyses reveal that there are fundamental structural differences between the two enzymes, and NfnII does not catalyze the NfnI bifurcating reaction. Instead, it exhibits non-bifurcating ferredoxin NADP oxidoreductase-type activity. NfnII is therefore proposed to be a bifunctional enzyme and also to catalyze a bifurcating reaction, although its third substrate, in addition to ferredoxin and NADP(H), is as yet unknown.

N-terminal structure of maize ferredoxin:NADP+ reductase determines recruitment into different thylakoid membrane complexes.

To adapt to different light intensities, photosynthetic organisms manipulate the flow of electrons through several alternative pathways at the thylakoid membrane. The enzyme ferredoxin:NADP(+) reductase (FNR) has the potential to regulate this electron partitioning because it is integral to most of these electron cascades and can associate with several different membrane complexes. However, the factors controlling relative localization of FNR to different membrane complexes have not yet been established. Maize (Zea mays) contains three chloroplast FNR proteins with totally different membrane association, and we found that these proteins have variable distribution between cells conducting predominantly cyclic electron transport (bundle sheath) and linear electron transport (mesophyll). Here, the crystal structures of all three enzymes were solved, revealing major structural differences at the N-terminal domain and dimer interface. Expression in Arabidopsis thaliana of maize FNRs as chimeras and truncated proteins showed the N-terminal determines recruitment of FNR to different membrane complexes. In addition, the different maize FNR proteins localized to different thylakoid membrane complexes on expression in Arabidopsis, and analysis of chlorophyll fluorescence and photosystem I absorbance demonstrates the impact of FNR location on photosynthetic electron flow.

Biochemical characterization of two recombinant ferredoxin reductases from Alcanivorax borkumensis SK2.

Alcanivorax borkumensis strain SK2 is a cosmopolitan oil-degrading oligotrophic marine γ-proteobacterium that exclusively uses petroleum hydrocarbons as sources of carbon and energy. Its ubiquity and unusual physiology suggest its global importance in the removal of hydrocarbons from polluted marine systems. The genome of A. borkumensis SK2 was recently sequenced. Two ferredoxin-nicotinamide adenine dinucleotide phosphate (NADPH) reductase genes (ABO_0145 and ABO_0203) have been annotated for this bacterium. In the present study, the expression, purification, and kinetic properties of these two genes were explored by constructing the prokaryotic expression vectors (pET21a) for the first time. Isopropyl ß-D-thiogalactoside (0.5 mM) was used for induction of exponentially growing cells (30 °C, overnight). Most of the proteins were expressed in inclusion body. Partial purification of recombinant enzymes was performed by ion-exchange chromatography on a DEAE-sepharose column using only one linear gradient of sodium chloride ranging between 0 and 500 mM. The recombinant enzymes displayed reductase activity, which was optimal at pH 6.0 and 45 °C. Ferredoxin-NADPH reductases exhibited several outstanding properties that made them excellent model proteins to address broad biological questions. This study serves as the basis for further investigations of the biotechnological potential of these enzymes.

Kinetic and optical biosensor study of adrenodoxin mutant AdxS112W displaying an enhanced interaction towards the cholesterol side chain cleavage enzyme (CYP11A1).

In mammals, steroid hormones are synthesized from cholesterol that is metabolized by the mitochondrial CYP11A1 system leading to pregnenolone. The reduction equivalents for this reaction are provided by NADPH, via a small electron transfer chain, consisting of adrenodoxin reductase (AdR) and adrenodoxin (Adx). The reaction partners are involved in a series of transient interactions to realize the electron transfer from NADPH to CYP11A1. Here, we compared the ionic strength effect on the AdR/Adx and Adx/CYP11A1 interactions for wild-type Adx and mutant AdxS112W. Using surface plasmon resonance measurements, stopped flow kinetic investigations and analyses of the product formation, we were able to obtain new insights into the mechanism of these interactions. The replacement of serine 112 by tryptophan was demonstrated to lead to a dramatically decreased k (off) rate of the Adx/CYP11A1 complex, resulting in a four-fold decreased K (d) value and indicating a much higher stability of the complex involving the mutant. Stopped flow analysis at various ionic strengths and in different mixing modes revealed that the binding of reduced Adx to CYP11A1 seems to display the limiting step for electron transfer to CYP11A1 with pre-reduced AdxS112W being much more efficient than wild-type Adx. Finally, the dramatic increase in pregnenolone formation at higher ionic strength using the mutant demonstrates that the interaction of CYP11A1 with Adx is the rate-limiting step in substrate conversion and that hydrophobic interactions may considerably improve this interaction and the efficiency of product formation. The data are discussed using published structural data of the complexes.

Ferredoxin-NADP reductase from the thermophilic hydrogen-oxidizing bacterium, Hydrogenobacter thermophilus TK-6.

The thermophilic, obligately chemolithoautotrophic hydrogen-oxidizing bacterium, Hydrogenobacter thermophilus TK-6, assimilates carbon dioxide via the reductive tricarboxylic acid cycle. Small iron-sulfur proteins, ferredoxins, play a central role as low-potential electron donors for this cycle. The fpr gene of this bacterium, encoding a putative ferredoxin-NADP(+) reductase (FNR, EC 1.18.1.2), was expressed in Escherichia coli, and the recombinant protein was purified to homogeneity. Unexpectedly, the monomeric Fpr protein contained one molecule of FMN as a prosthetic group, although FNRs from other organisms are known to contain FAD. The FMN-containing Fpr was shown to be a bona fide FNR that catalyzes a reversible redox reaction between NADP(+)/NADPH and ferredoxins.

Impeded electron transfer from a pathogenic FMN domain mutant of methionine synthase reductase and its responsiveness to flavin supplementation.

Methionine synthase reductase (MSR) is a diflavin oxidoreductase that transfers electrons from NADPH to oxidized cobalamin and plays a vital role in repairing inactive cobalamin-dependent methionine synthase. MSR deficiency is a recessive genetic disorder affecting folate and methionine metabolism and is characterized by elevated levels of plasma homocysteine. In this study, we have examined the molecular basis of MSR dysfunction associated with a patient mutation, A129T, which is housed in the FMN binding domain and is adjacent to a cluster of conserved acidic residues found in diflavin oxidoreductases. We show that the substitution of alanine with threonine destabilizes FMN binding without affecting the NADPH coenzyme specificity or affinity, indicating that the mutation's effects may be confined to the FMN module. The A129T MSR mutant transfers electrons to ferricyanide as efficiently as wild type MSR but the rate of cytochrome c, 2,6-dichloroindophenol, and menadione reduction is decreased 10-15 fold. The mutant is depleted in FMN and reactivates methionine synthase with 8% of the efficiency of wild type MSR. Reconstitution of A129T MSR with FMN partially restores its ability to reduce cytochrome c and to reactivate methionine synthase. Hydrogen-deuterium exchange mass spectrometric studies localize changes in backbone amide exchange rates to peptides in the FMN-binding domain. Together, our results reveal that the primary biochemical penalty associated with the A129T MSR mutant is its lower FMN content, provide insights into the distinct roles of the FAD and FMN centers in human MSR for delivering electrons to various electron acceptors, and suggest that patients harboring the A129T mutation may be responsive to riboflavin therapy.

Toxoplasma gondii ferredoxin-NADP+ reductase: Role of ionic interactions in stabilization of native conformation and structural cooperativity.

The apicoplast and the proteins present therein are parasite-specific targets for chemotherapy of apicomplexan parasites. Ferredoxin-NADP(+) reductase (FNR) is an important enzyme present in the apicoplast of Toxoplasma gondii that operates as a general electron switch at the bifurcation step of many different electron transfer pathways. In spite of its importance as drug target not much structural information on the enzyme is available. Using fluorescence and CD spectroscopy in combination with enzyme activity measurement and size exclusion chromatography, we studied the pH-dependent changes in structural and functional properties and interdomain interactions in recombinant Toxoplasma gondii ferredoxin-NADP(+) reductase (TgFNR) to understand the interactions responsible for stabilization of native conformation and modulation of functional activity of the enzyme. Under physiological conditions, the recombinant TgFNR is stabilized in an open conformation. The open conformation of the enzyme was found to be essential for its optimum functioning, as induction of compactness/rigidity by modulation of pH, leads to decrease in the functional activity. In native conformation, strong interactions exist between the NADP(+)- and FAD-binding domains thus making the enzyme a structurally cooperative molecule. Under acidic conditions (pH about 4), the interdomain interactions present in native TgFNR were lost and the enzyme became structurally noncooperative. The pH-induced structural alterations in the NADP(+) binding domain, more precisely compaction of the conformation lead to its stabilization against thermal denaturation. The studies demonstrate the significance of electrostatic interactions both in stabilization of native conformation and maintenance of structural cooperativity in TgFNR.

Crystal structures of Leptospira interrogans FAD-containing ferredoxin-NADP+ reductase and its complex with NADP+.

BACKGROUND: Ferredoxin-NADP(H) reductases (FNRs) are flavoenzymes that catalyze the electron transfer between NADP(H) and the proteins ferredoxin or flavodoxin. A number of structural features distinguish plant and bacterial FNRs, one of which is the mode of the cofactor FAD binding. Leptospira interrogans is a spirochaete parasitic bacterium capable of infecting humans and mammals in general. Leptospira interrogans FNR (LepFNR) displays low sequence identity with plant (34% with Zea mays) and bacterial (31% with Escherichia coli) FNRs. However, LepFNR contains all consensus sequences that define the plastidic class FNRs. RESULTS: The crystal structures of the FAD-containing LepFNR and the complex of the enzyme with NADP+, were solved and compared to known FNRs. The comparison reveals significant structural similarities of the enzyme with the plastidic type FNRs and differences with the bacterial enzymes. Our small angle X-ray scattering experiments show that LepFNR is a monomeric enzyme. Moreover, our biochemical data demonstrate that the LepFNR has an enzymatic activity similar to those reported for the plastidic enzymes and that is significantly different from bacterial flavoenzymes, which display lower turnover rates. CONCLUSION: LepFNR is the first plastidic type FNR found in bacteria and, despite of its low sequence similarity with plastidic FNRs still displays high catalytic turnover rates. The typical structural and biochemical characteristics of plant FNRs unveiled for LepFNR support a notion of a putative lateral gene transfer which presumably offers Leptospira interrogans evolutionary advantages. The wealth of structural information about LepFNR provides a molecular basis for advanced drugs developments against leptospirosis.

Crystallization and preliminary X-ray diffraction studies of ferredoxin reductase from Leptospira interrogans.

Ferredoxin-NADP+ reductase (FNR) is an FAD-containing enzyme that catalyzes electron transfer between NADP(H) and ferredoxin. Here, results are reported of the recombinant expression, purification and crystallization of FNR from Leptospira interrogans, a parasitic bacterium of animals and humans. The L. interrogans FNR crystals belong to a primitive monoclinic space group and diffract to 2.4 angstroms resolution at a synchrotron source.

Human methionine synthase reductase is a molecular chaperone for human methionine synthase.

Sustained activity of mammalian methionine synthase (MS) requires MS reductase (MSR), but there have been few studies of the interactions between these two proteins. In this study, recombinant human MS (hMS) and MSR (hMSR) were expressed in baculovirus-infected insect cells and purified to homogeneity. hMSR maintained hMS activity at a 1:1 stoichiometric ratio with a K(act) value of 71 nM. Escherichia coli MS, however, was not activated by hMSR. Moreover, hMS was not significantly active in the presence of E. coli flavodoxin and flavodoxin reductase, which maintain the activity of E. coli MS. These results indicate that recognition of MS by their reductive partners is very strict, despite the high homology between MS from different species. The effects of hMSR on the formation of hMS holoenzyme also were examined by using crude extracts of baculovirus-infected insect cells containing hMS apoenzyme (apoMS). In the presence of MSR and NADPH, holoenzyme formation from apoMS and methylcobalamin was significantly enhanced. The observed stimulation is shown to be due to stabilization of human apoMS in the presence of MSR. Apoenzyme alone is quite unstable at 37 degrees C. MSR also is able to reduce aquacobalamin to cob(II)alamin in the presence of NADPH, and this reduction leads to stimulation of the conversion of apoMS and aquacobalamin to MS holoenzyme. Based on these findings, we propose that MSR serves as a special chaperone for hMS and as an aquacobalamin reductase, rather than acting solely in the reductive activation of MS.

Analysis of the nearly identical morpholine monooxygenase-encoding mor genes from different Mycobacterium strains and characterization of the specific NADH : ferredoxin oxidoreductase of this cytochrome P450 system.

Cloning and sequencing of the morABC operon region revealed the genes encoding the three components of a cytochrome P450 monooxygenase, which is required for the degradation of the N-heterocycle morpholine by Mycobacterium sp. strain HE5. The cytochrome P450 (P450(mor)) and the Fe(3)S(4) ferredoxin (Fd(mor)), encoded by morA and morB, respectively, have been characterized previously, whereas no evidence has hitherto been obtained for a specifically morpholine-induced reductase, which would be required to support the activity of the P450(mor) system. Analysis of the mor operon has now revealed the gene morC, encoding the ferredoxin reductase of this morpholine monooxygenase. The genes morA, morB and morC were identical to the corresponding genes from Mycobacterium sp. strain RP1. Almost identical mor genes in Mycobacterium chlorophenolicum PCP-1, in addition to an inducible cytochrome P450, pointing to horizontal gene transfer, were now identified. No evidence for a circular or linear plasmid was found in Mycobacterium sp. strain HE5. Analysis of the downstream sequences of morC revealed differences in this gene region between Mycobacterium sp. strain HE5 and Mycobacterium sp. strain RP1 on the one hand, and M. chlorophenolicum on the other hand, indicating insertions or deletions after recombination. Downstream of the mor genes, the gene orf1', encoding a putative glutamine synthetase, was identified in all studied strains. The gene morC of Mycobacterium sp. strain HE5 was heterologously expressed. The purified recombinant protein FdR(mor) was characterized as a monomeric 44 kDa protein, being a strictly NADH-dependent, FAD-containing reductase. The K(m) values of FdR(mor) for the substrate NADH (37.7 +/- 4.1 microM) and the artificial electron acceptors potassium ferricyanide (14.2 +/- 1.1 microM) and cytochrome c (28.0 +/- 3.6 microM) were measured. FdR(mor) was shown to interact functionally with its natural redox partner, the Fe(3)S(4) protein Fd(mor), and with the Fe(2)S(2) protein adrenodoxin, albeit with a much lower efficiency, but not with spinach ferredoxin. In contrast, adrenodoxin reductase, the natural redox partner of adrenodoxin, could not use Fd(mor) in activity assays. These results indicated that FdR(mor) can utilize different ferredoxins, but that Fd(mor) requires the specific NADH : ferredoxin oxidoreductase FdR(mor) from the P450(mor) system for efficient catalytic function.

Towards a new interaction enzyme:coenzyme.

Ferredoxin-NADP(+) reductase catalyses NADP(+) reduction, being specific for NADP(+)/H. To understand coenzyme specificity determinants and coenzyme specificity reversion, mutations at the NADP(+)/H pyrophosphate binding and of the C-terminal regions have been simultaneously introduced in Anabaena FNR. The T155G/A160T/L263P/Y303S mutant was produced. The mutated enzyme presents similar k(cat) values for NADPH and NADH, around 2.5 times slower than that reported for WT FNR with NADPH. Its K(m) value for NADH decreased 20-fold with regard to WT FNR, whereas the K(m) for NADPH remains similar. The combined effect is a much higher catalytic efficiency for NAD(+)/H, with a minor decrease of that for NADP(+)/H. In the mutated enzyme, the specificity for NADPH versus NADH has been decreased from 67,500 times to only 12 times, being unable to discriminate between both coenzymes. Additionally, giving the role stated for the C-terminal Tyr in FNR, its role in the energetics of the FAD binding has been analysed.

Kinetic and binding studies with purified recombinant proteins ferredoxin reductase, ferredoxin and cytochrome P450 comprising the morpholine mono-oxygenase from Mycobacterium sp. strain HE5.

The P450mor system from Mycobacterium sp. strain HE5, supposed to catalyse the hydroxylation of different N-heterocycles, is composed of three components: ferredoxin reductase (FdRmor), Fe3S4 ferredoxin (Fdmor) and cytochrome P450 (P450mor). In this study, we purified Fdmor and P450mor as recombinant proteins as well as FdRmor, which has been isolated previously. Kinetic investigations of the redox couple FdRmor/Fdmor revealed a 30-fold preference for the NADH-dependent reduction of nitroblue tetrazolium (NBT) and an absolute requirement for Fdmor in this reaction, compared with the NADH-dependent reduction of cytochrome c. The quite low Km (5.3 +/- 0.3 nm) of FdRmor for Fdmor, measured with NBT as the electron acceptor, indicated high specificity. The addition of sequences providing His-tags to the N- or C-terminus of Fdmor did not significantly alter kinetic parameters, but led to competitive background activities of these fusion proteins. Production of P450mor as an N-terminal His-tag fusion protein enabled the purification of this protein in its spectral active form, which has previously not been possible for wild-type P450mor. The proposed substrates morpholine, piperidine or pyrrolidine failed to produce substrate-binding spectra of P450mor under any conditions. Pyridine, metyrapone and different azole compounds generated type II binding spectra and the Kd values determined for these substances suggested that P450mor might have a preference for more bulky and/or hydrophobic molecules. The purified recombinant proteins FdRmor, Fdmor and P450mor were used to reconstitute the homologous P450-containing mono-oxygenase, which was shown to convert morpholine.

Purification and characterization of ferredoxin-NADP+ reductase encoded by Bacillus subtilis yumC.

From Bacillus subtilis cell extracts, ferredoxin-NADP+ reductase (FNR) was purified to homogeneity and found to be the yumC gene product by N-terminal amino acid sequencing. YumC is a approximately 94-kDa homodimeric protein with one molecule of non-covalently bound FAD per subunit. In a diaphorase assay with 2,6-dichlorophenol-indophenol as electron acceptor, the affinity for NADPH was much higher than that for NADH, with Km values of 0.57 microM vs >200 microM. Kcat values of YumC with NADPH were 22.7 s(-1) and 35.4 s(-1) in diaphorase and in a ferredoxin-dependent NADPH-cytochrome c reduction assay, respectively. The cell extracts contained another diaphorase-active enzyme, the yfkO gene product, but its affinity for ferredoxin was very low. The deduced YumC amino acid sequence has high identity to that of the recently identified Chlorobium tepidum FNR. A genomic database search indicated that there are more than 20 genes encoding proteins that share a high level of amino acid sequence identity with YumC and which have been annotated variously as NADH oxidase, thioredoxin reductase, thioredoxin reductase-like protein, etc. These genes are found notably in gram-positive bacteria, except Clostridia, and less frequently in archaea and proteobacteria. We propose that YumC and C. tepidum FNR constitute a new group of FNR that should be added to the already established plant-type, bacteria-type, and mitochondria-type FNR groups.

The oxidant-responsive diaphorase of Rhodobacter capsulatus is a ferredoxin (flavodoxin)-NADP(H) reductase.

Challenge of Rhodobacter capsulatus cells with the superoxide propagator methyl viologen resulted in the induction of a diaphorase activity identified as a member of the ferredoxin (flavodoxin)-(reduced) nicotinamide adenine dinucleotide phosphate (NADP(H)) reductase (FPR) family by N-terminal sequencing. The gene coding for Rhodobacter FPR was cloned and expressed in Escherichia coli. Both native and recombinant forms of the enzyme were purified to homogeneity rendering monomeric products of approximately 30 kDa with essentially the same spectroscopic and kinetic properties. They were able to bind and reduce Rhodobacter flavodoxin (NifF) and to mediate typical FPR activities such as the NADPH-driven diaphorase and cytochrome c reductase.

Purification and characterization of carbazole 1,9a-dioxygenase, a three-component dioxygenase system of Pseudomonas resinovorans strain CA10.

The carbazole 1,9a-dioxygenase (CARDO) system of Pseudomonas resinovorans strain CA10 consists of terminal oxygenase (CarAa), ferredoxin (CarAc), and ferredoxin reductase (CarAd). Each component of CARDO was expressed in Escherichia coli strain BL21(DE3) as a native form (CarAa) or a His-tagged form (CarAc and CarAd) and was purified to apparent homogeneity. CarAa was found to be trimeric and to have one Rieske type [2Fe-2S] cluster and one mononuclear iron center in each monomer. Both His-tagged proteins were found to be monomeric and to contain the prosthetic groups predicted from the deduced amino acid sequence (His-tagged CarAd, one FAD and one [2Fe-2S] cluster per monomer protein; His-tagged CarAc, one Rieske type [2Fe-2S] cluster per monomer protein). Both NADH and NADPH were effective as electron donors for His-tagged CarAd. However, since the k(cat)/K(m) for NADH is 22.3-fold higher than that for NADPH in the 2,6-dichlorophenolindophenol reductase assay, NADH was supposed to be the physiological electron donor of CarAd. In the presence of NADH, His-tagged CarAc was reduced by His-tagged CarAd. Similarly, CarAa was reduced by His-tagged CarAc, His-tagged CarAd, and NADH. The three purified proteins could reconstitute the CARDO activity in vitro. In the reconstituted CARDO system, His-tagged CarAc seemed to be indispensable for electron transport, while His-tagged CarAd could be replaced by some unrelated reductases.

Differences in the efficiency of reductive activation of methionine synthase and exogenous electron acceptors between the common polymorphic variants of human methionine synthase reductase.

Methionine synthase reductase (MSR) catalyzes the conversion of the inactive form of human methionine synthase to the active state of the enzyme. This reaction is of paramount physiological importance since methionine synthase is an essential enzyme that plays a key role in the methionine and folate cycles. A common polymorphism in human MSR has been identified (66A --> G) that leads to replacement of isoleucine with methionine at residue 22 and has an allele frequency of 0.5. Another polymorphism is 524C --> T, which leads to the substitution of serine 175 with leucine, but its allele frequency is not known. The I22M polymorphism is a genetic determinant for mild hyperhomocysteinemia, a risk factor for cardiovascular disease. In this study, we have examined the kinetic properties of the M22/S175 and I22/S175 and the I22/L175 and I22/S175 pairs of variants. EPR spectra of the semiquinone forms of variants I22/S175 and M22/S175 are indistinguishable and exhibit an isotropic signal at g = 2.00. In addition, the electronic absorption and reduction stoichiometries with NADPH are identical in these variants. Significantly, the variants activate methionine synthase with the same V(max); however, a 3-4-fold higher ratio of MSR to methionine synthase is required to elicit maximal activity with the M22/S175 and I22/L175 variant versus the I22/S175 enzyme. Differences are also observed between the variants in the efficacies of reduction of the artificial electron acceptors: ferricyanide, 2,6-dichloroindophenol, 3-acetylpyridine adenine dinucleotide phosphate, menadione, and the anticancer drug doxorubicin. These results reveal differences in the interactions between the natural and artificial electron acceptors and MSR variants in vitro, which are predicted to result in less efficient reductive repair of methionine synthase in vivo.

Ferredoxin-NADP+ reductase and ferredoxin of the protozoan parasite Toxoplasma gondii interact productively in Vitro and in Vivo.

Toxoplasma gondii possesses an apicoplast-localized, plant-type ferredoxin-NADP(+) reductase. We have cloned a [2Fe-2S] ferredoxin from the same parasite to investigate the interplay of the two redox proteins. A detailed characterization of the two purified recombinant proteins, particularly as to their interaction, has been performed. The two-protein complex was able to catalyze electron transfer from NADPH to cytochrome c with high catalytic efficiency. The redox potential of the flavin cofactor (FAD/FADH(-)) of the reductase was shown to be more positive than that of the NADP(+)/NADPH couple, thus favoring electron transfer from NADPH to yield reduced ferredoxin. The complex formation between the reductase and ferredoxins from various sources was studied both in vitro by several approaches (enzymatic activity, cross-linking, protein fluorescence quenching, affinity chromatography) and in vivo by the yeast two-hybrid system. Our data show that the two proteins yield an active complex with high affinity, strongly suggesting that the two proteins of T. gondii form a physiological redox couple that transfers electrons from NADPH to ferredoxin, which in turn is used by some reductive biosynthetic pathway(s) of the apicoplast. These data provide the basis for the exploration of this redox couple as a drug target in apicomplexan parasites.

The complete purification and characterization of three forms of ferredoxin-NADP(+) oxidoreductase from a thermophilic cyanobacterium Synechococcus elongatus.

The petH gene, encoding ferredoxin-NADP(+) oxidoreductase (FNR), was isolated from a thermophilic cyanobacterium, Synechococcus elongatus (the same strain as Thermosynechococcus elongatus). The petH gene of S. elongatus was a single copy gene, and the N-terminal region of PetH showed a sequence similarity to the CpcD-phycobilisome linker polypeptide. The amino acid sequence of the catalytic domains of PetH was markedly similar to those from mesophilic cyanobacterial PetH and higher plant FNR. The enzymatically active FNR protein was purified to homogeneity from S. elongatus as three forms corresponding to the 45-kDa form retaining the CpcD-like domain, the 34-kDa form lacking the CpcD-like domain, and the 78-kDa complex with phycocyanin. The FNR in the 78-kDa complex was tolerant to proteolytic cleavage. However, the dissociation of phycocyanin from the 78-kDa complex induced to specific proteolysis between the CpcD-like domain and the FAD-binding domain to give rise to the 34-kDa form of FNR. The enzymatic activity of the 45-kDa form was thermotolerant, but the 45-kDa form readily aggregated under the storage at -30 degrees C. These results suggest that the association with phycocyanin via CpcD-like domain gives remarkable stability to S. elongatus FNR.

Purification and characterization of ferredoxin-NAD(P)(+) reductase from the green sulfur bacterium Chlorobium tepidum.

Ferredoxin-NAD(P)(+) reductase [EC 1.18.1.3, 1.18.1.2] was isolated from the green sulfur bacterium Chlorobium tepidum and purified to homogeneity. The molecular mass of the subunit is 42 kDa, as deduced by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The molecular mass of the native enzyme is approximately 90 kDa, estimated by gel-permeation chromatography, and is thus a homodimer. The enzyme contains one FAD per subunit and has absorption maxima at about 272, 385, and 466 nm. In the presence of ferredoxin (Fd) and reaction center (RC) complex from C. tepidum, it efficiently catalyzes photoreduction of both NADP(+) and NAD(+). When concentrations of NADP(+) exceeded 10 microM, NADP(+) photoreduction rates decreased with increased concentration. The inhibition by high concentrations of substrate was not observed with NAD(+). It also reduces 2,6-dichlorophenol-indophenol (DPIP) and molecular oxygen with either NADPH or NADH as efficient electron donors. It showed NADPH diaphorase activity about two times higher than NADH diaphorase activity in DPIP reduction assays at NAD(P)H concentrations less than 0.1 mM. At 0.5 mM NAD(P)H, the two activities were about the same, and at 1 mM, the former activity was slightly lower than the latter.