Plasmodium-specific basic amino acid residues important for the interaction with ferredoxin on the surface of ferredoxin-NADP+ reductase

Plasmodium-specific basic amino acid residues important for the interaction with ferredoxin on... Abstract The malaria parasite (Plasmodium falciparum) possesses a plastid-derived, essential organelle called the apicoplast, which contains a redox system comprising plant-type ferredoxin (Fd) and Fd-NADP+ reductase (FNR). This system supplies reducing power for the crucial metabolic pathways in this organelle. Electron transfer between P. falciparum Fd (PfFd) and FNR (PfFNR) is performed with higher affinity and specificity than that of plant Fd and FNR. To investigate the mechanism for such superior protein–protein interaction, we searched for the Fd interaction sites on the surface of PfFNR. Basic amino acid residues on the FAD binding side of PfFNR were comprehensively substituted to acidic amino acids by site-directed mutagenesis. Kinetic analysis of electron transfer to PfFd and plant Fds, physical binding to immobilized PfFd and thermodynamics of the PfFd binding using these PfFNR mutants revealed that several basic amino acid residues including those in Plasmodium-specific insertion region are important for the interaction with PfFd. Majority of these basic residues are Plasmodium-specific and not conserved among plant and cyanobacteria FNRs. These results suggest that the interaction mode of Fd and FNR is diverged during evolution so that PfFd: PfFNR interaction meets the physiological requirement in the cells of Plasmodium species. ferredoxin, ferredoxin-NADP+, reductase, malaria parasite, plastid evolution, protein–protein interaction Protozoan parasites of the phylum Apicomplexa, including Plasmodium sp. (the causative agent of malaria) and Toxoplasma sp., contain a non-photosynthetic plastid organelle called the apicoplast (1), which was acquired by secondary endosymbiosis of algae (2). The apicoplast was shown to be essential for the parasite’s survival (3, 4) and therefore considered as an ideal drug target without distressing the animal host. The vital function of the apicoplast has been investigated; the biosynthesis of isoprenoids was shown to be the only essential role in the erythrocytic life stage (5), while the biosynthesis of fatty acids, Fe-S clusters and hemes may be essential in other stages such as the liver stage or the mosquito stage (6). We previously reported the cloning and characterization of plant-type ferredoxin (Fd) and Fd-NADP+ reductase (FNR) from Plasmodium falciparum (human malaria parasite), and proposed that they constitute a redox system which supplies reducing power that drives the biosynthetic reactions in the apicoplast (7). In higher plants, Fd is mainly reduced by the photosynthetic electron transport chain and donates reducing equivalents to FNR and other Fd-dependent enzymes involved in various metabolic and regulatory reactions in the plastid (8–10). In non-photosynthetic tissues such as roots, these Fd-dependent enzymes are also functioning, but the reduction of Fd is catalysed by FNR, using NADPH generated through the oxidative pentose phosphate cycle (11). These discrete redox cascades are conducted by the combination of genetically distinct isoforms of Fd and FNR present differentially in photosynthetic and non-photosynthetic tissues (12). By analogy to the non-photosynthetic type in plants, a redox cascade of NADPH–FNR–Fd has been considered to provide reducing power for the crucial Fd-dependent metabolisms in the apicoplasts (5). The involvement of apicoplast Fd has been indicated in the reactions of an Fe-S cluster assembly pathway (13), isoprenoid biosynthesis pathway (14, 15), fatty acid desaturation (16) and heme oxygenation (17). Because this plant-type redox system is not present in the mammalian host, it would represent a promising drug target to combat malaria, for instance, by developing substances which inhibit the interaction between Fd and FNR from malaria parasites (18). The amino acid sequence of P. falciparum Fd (PfFd) (Supplementary Fig. S1A) shares about 50% homology with plant Fds (16, 19), and its backbone structure, as solved by X-ray crystallography, closely resembles to those of plant Fds (7). On the other hand, P. falciparum FNR (PfFNR) (Supplementary Fig. S1B) shares lower homology (20–30%) with plant FNRs, displaying large insertions and deletions specific to Plasmodium sp. (20) which appear to be responsible for the unique disordered surface structures shown by its crystal structure analysis (21). In a reconstituted assay system, electron transfer between PfFd and PfFNR is performed with higher affinity for the Fd (about 1 μM of Km) (7) than that of plant Fd: FNR combination (3–4 μM of Km). In addition, PfFNR transfers electrons preferentially to PfFd with much higher (several to 10 times) affinity over plant Fds (7), indicating their advantageous interaction. Since crystal structures of PfFd (7) and PfFNR (21) have been determined, several studies have addressed the structure–function characteristics of PfFNR (21–23), but the molecular interaction between PfFd and PfFNR has not been addressed well, except for our previous study (24). Fd and FNR form 1:1 complex for the efficient electron transfer between their prosthetic groups of the 2Fe-2S cluster and FAD, respectively. X-ray crystal structures of the complexes of Fd and FNR from leaf (25) and root (26) in higher plants and from cyanobacteria (Anabaena PCC7119) (27) have been reported, clarifying the sites involved in the complex formation between the two protein molecules. Unexpectedly, the interaction mode of Fd and FNR, such as the binding surface area and the orientation of Fd relative to FNR, is largely different between the Fd–FNR pairs of photosynthetic (leaf) type and non-photosynthetic (root) type (26). These distinct Fd–FNR interaction modes between the two types of the complexes may be due to the optimization for their efficiency in photosynthetic and non-photosynthetic (heterotrophic) electron transfer cascades, which are opposite in terms of the direction of electron flow (26) as described above. The Fd-FNR interaction mode of the Anabaena complex which physiologically operates both directions of electron flow is also different from either of the plant complexes (27). However, there is a common feature that multiple salt bridges mostly between acidic residues of Fd and basic residues of FNR stabilize the complex formation; there are five salt bridges in the leaf complex, three in the root complex and two in the Anabaena complex. The amino acid residues involved in these salt bridges are mostly conserved among Fd species, but largely different among FNRs. Thus, FNR interacts with Fd using the amino acid residues specific to each FNR species, which determines the iso-type specific orientation of Fd relative to FNR (26). So far, the 3D structure of the PfFd–PfFNR complex is not available, but several acidic amino acid residues on PfFd (e.g. D26, E29, E34, D65 and E66) were shown to be important for the interaction with PfFNR by the NMR chemical shift perturbation analysis combined with site-directed mutagenesis to neutral amino acids (24). Therefore, the electrostatic interaction between these acidic residues in PfFd and certain basic residues in PfFNR is expected to be important for the complex formation. In this study, we found that several basic amino acid residues on the FAD binding side of PfFNR are important for the interaction with Pf Fd, and that majority of these FNR residues are specific to Plasmodium sp. and not conserved among plants and cyanobacteria. Materials and Methods Site-directed mutagenesis of PfFNR and preparation of recombinant proteins Cloning and preparation of PfFNR, PfFd (wild-type and mutants) and maize Fds were described previously (7, 24, 28). For the construction of PfFNR mutants, the QuikChange site-directed mutagenesis kit (Agilent Technologies, USA) was used according to the manufacturer’s instructions. The synthetic oligonucleotides used for the mutagenesis are shown in Supplementary Table S1. The mutation sites and the sequence integrity of the entire coding region of PfFNR were confirmed by DNA sequencing. Enzymatic analysis Enzyme activity of PfFNR was measured using a grating microplate reader (model SH-1000 Lab, CORONA, Japan). The activity of NADPH-dependent electron transfer from FNR to Fd was measured using cytochrome c (cyt c) as a final electron acceptor as described previously (28) except that the concentrations of PfFNR and NADPH were increased to 50 nM and 500 μM, respectively. Diaphorase activity of FNR with DCPIP as an electron acceptor was measured as described previously (28) except that the concentration of PfFNR was lowered to 25 nM and NaCl at 100 mM was included. Pull-down assay Immobilized PfFd was obtained by coupling PfFd to CNBr-activated-Sepharose 4B (GE Healthcare Bio-Science, USA), according to the manufacturer’s directions. PfFNR mutants (0.20 nmol) were incubated with 20 μl of the immobilized PfFd resin (containing 4.5 nmol of PfFd) in the solution of 50 mM Tris–HCl, pH 7.5, 100 mM NaCl, and the resin was washed three times with the same solution and centrifuged. The resulting resin pellet was extracted with 50 μl of SDS sample buffer, and 10 μl of the extract was analysed by 12.5% SDS PAGE. Isothermal titration calorimetry (ITC) Protein samples were dialyzed against 50 mM Tris–HCl, pH 7.5, 100 mM NaCl, and degassed for 3 min before being loaded into the calorimeter. Calorimetric experiments were performed with an Auto-iTC200 instrument (GE Healthcare Biosciences) at 298 K. In the injection syringe, 500 μM PfFd was titrated into 50 μM wild-type or mutant PfFNR in the ITC cell. Titration experiments consisted of 38 injections spaced at intervals of 150 s. The injection volume was 1.0 μl and the cell was continuously stirred at 1000 rpm. Thermodynamic parameters of the complex formation between Fd and FNR were obtained as described previously (29). Results Preparation of site-directed mutants of Pf FNR Basic amino acid residues on the molecular surface of the FAD binding side of PfFNR (Fig. 1) were comprehensively mutated to acidic residues. Eleven mutants were prepared; two double mutants (K73E/H75D and H83D/H86D) and one triple mutant (H93D/K95E/K96E) in the Plasmodium-specific insertion region (shown as dotted line in Fig. 1), and eight single mutants (R98E, H157D, H286D, K287E, R290E, K298E, K308E and K309E) in other regions. UV-visible spectra of these 11 mutants showed no significant difference from that of wild-type PfFNR (7), indicating that the holo-form enzymes containing FAD were produced. SDS PAGE analysis of the mutant PfFNRs (Fig. 2) showed similar mobility on the gel except that K73E/H75D mutant showed slightly slower mobility and H93D/K95E/K96E mutant appeared to be mostly cleaved during storage and/or during SDS PAGE (full-size FNR protein fragment was observed in the fresh preparation of this mutant by MALDI-TOF mass analysis as shown in Supplementary Fig. S2). Therefore, PfFNR mutants other than the triple mutant were used for the further analyses. Fig. 1 View largeDownload slide Molecular surface of PfFNR with basic residues mutated in this study. Mutated basic residues are shown as sticks with mesh, and FAD and NADPH-analogue, adenosine 2′, 5′-diphosphate (A2P), are shown as sticks on the X-ray crystal structure of PfFNR (PDB code 2OK7). There are five disordered surface regions in the structure which could not be presented as surface model, and among them, the region from N61 to I94 in a Plasmodium-specific long insertion is shown as dotted line. The number of * and + depicted beside the residue numbers stand for the extent of the changes in the Km value and the physical Fd-binding, respectively, analysed in this study. ND stands for ‘not determined’. Fig. 1 View largeDownload slide Molecular surface of PfFNR with basic residues mutated in this study. Mutated basic residues are shown as sticks with mesh, and FAD and NADPH-analogue, adenosine 2′, 5′-diphosphate (A2P), are shown as sticks on the X-ray crystal structure of PfFNR (PDB code 2OK7). There are five disordered surface regions in the structure which could not be presented as surface model, and among them, the region from N61 to I94 in a Plasmodium-specific long insertion is shown as dotted line. The number of * and + depicted beside the residue numbers stand for the extent of the changes in the Km value and the physical Fd-binding, respectively, analysed in this study. ND stands for ‘not determined’. Fig. 2 View largeDownload slide SDS PAGE analysis of purified Pf FNRs. Wild-type and mutant PfFNR proteins (about 1 μg each) were analysed by 12.5% SDS PAGE and stained with coomassie brilliant blue. Triple mutant of H93D/K95E/K96E was mostly cleaved as detailed in the text. Partially cleaved products (20–24 kDa) were also observed with other FNRs. Fig. 2 View largeDownload slide SDS PAGE analysis of purified Pf FNRs. Wild-type and mutant PfFNR proteins (about 1 μg each) were analysed by 12.5% SDS PAGE and stained with coomassie brilliant blue. Triple mutant of H93D/K95E/K96E was mostly cleaved as detailed in the text. Partially cleaved products (20–24 kDa) were also observed with other FNRs. Kinetic analysis of electron transfer from Pf FNR mutants to Pf Fd The activity of NADPH-dependent electron transfer from the 10 PfFNR mutants to PfFd was measured (Table I). The PfFNR mutants except for H286D and H83D/H86D showed significant increases in the Km for PfFd as compared to wild-type PfFNR, indicating that the charge reversal mutation of only one or two residues in the eight PfFNR mutants considerably affects the interaction with PfFd. The extent of the effect is variable (from 4 to 11 times of Km value) depending on the mutation site, and the average Km value increased in the order of K73E/H75D, K309E, K287E, K298E, R290E, H157D, R98E and K308E PfFNRs. H286D mutant did not show significant change in the Km but exhibited a large decrease in the kcat (2.9 s−1) as compared to other PfFNRs (10–32 s−1) (Table I). H286 is located in the vicinity of the NADPH binding site (A2P, an NADPH analogue, is shown in Fig. 1) and its hydrogen-bonding ability was suggested to be important for the binding of NADPH (23). Measurement of NADPH-dependent diaphorase activity of H286D mutant showed a large decrease in the affinity for NADPH (several times increase in the Km; Table I) as compared to wild-type PfFNR, which mostly explains the observed reduction in the electron transfer (cyt c reduction) activity performed at 500 μM NADPH. For other FNRs analyzed, the Km values for NADPH were much lower than 500 μM NADPH, and the kcat values for the diaphorase activity were more than 2.4 times higher than those of cyt c reduction, indicating that the FNR reduction by NADPH is not a rate-limiting step in the cyt c reduction assay under the experimental conditions in this study. Table I. Steady-state kinetic parameters of wild-type and mutated PfFNRs in the reactions of NADPH-dependent cyt c reduction using PfFd and of NADPH-dependent diaphorase assay using DCPIP PfFNR  NADPH-dependent cyt c reduction   NADPH-dependent diaphorase assay   Km for PfFd (μM)  kcat (s−1)  Km for NADPH (μM)  kcat (s−1)  Wild-type  1.3±0.5  25±1  110±40  59±6  R98E  11±3  19±4  74±21  81±17  H157D  9.5±4.4  10±1  59±11  56±11  H286D  0.96±0.70  2.9±0.3  760±450  20±11  K287E  7.9±1.9  21±2  ND  ND  R290E  8.9±5.2  14±4  ND  ND  K298E  8.2±2.2  32±7  ND  ND  K308E  15±6  22±5  ND  ND  K309E  6.2±3.5  15±3  67±12  88±20  K73E/H75D  6.0±2.8  27±10  ND  ND  H83D/H86D  3.1±1.9  26±8  ND  ND  PfFNR  NADPH-dependent cyt c reduction   NADPH-dependent diaphorase assay   Km for PfFd (μM)  kcat (s−1)  Km for NADPH (μM)  kcat (s−1)  Wild-type  1.3±0.5  25±1  110±40  59±6  R98E  11±3  19±4  74±21  81±17  H157D  9.5±4.4  10±1  59±11  56±11  H286D  0.96±0.70  2.9±0.3  760±450  20±11  K287E  7.9±1.9  21±2  ND  ND  R290E  8.9±5.2  14±4  ND  ND  K298E  8.2±2.2  32±7  ND  ND  K308E  15±6  22±5  ND  ND  K309E  6.2±3.5  15±3  67±12  88±20  K73E/H75D  6.0±2.8  27±10  ND  ND  H83D/H86D  3.1±1.9  26±8  ND  ND  The kcat values are expressed as the numbers per electron equivalent. The values are mean ± SD of at least three independent measurements. ‘ND’ stands for ‘Not determined’. As for the effect of mutations near the NADPH binding site, the diaphorase activity of H286D mutant at the same NADPH concentration for the cyt c reduction assay (500 μM) was considerably low (9.5 s−1), while the activities of K287E and R290E mutants (53 and 65 s−1, respectively) was comparable to that of wild-type PfFNR (63 s−1). Table I. Steady-state kinetic parameters of wild-type and mutated PfFNRs in the reactions of NADPH-dependent cyt c reduction using PfFd and of NADPH-dependent diaphorase assay using DCPIP PfFNR  NADPH-dependent cyt c reduction   NADPH-dependent diaphorase assay   Km for PfFd (μM)  kcat (s−1)  Km for NADPH (μM)  kcat (s−1)  Wild-type  1.3±0.5  25±1  110±40  59±6  R98E  11±3  19±4  74±21  81±17  H157D  9.5±4.4  10±1  59±11  56±11  H286D  0.96±0.70  2.9±0.3  760±450  20±11  K287E  7.9±1.9  21±2  ND  ND  R290E  8.9±5.2  14±4  ND  ND  K298E  8.2±2.2  32±7  ND  ND  K308E  15±6  22±5  ND  ND  K309E  6.2±3.5  15±3  67±12  88±20  K73E/H75D  6.0±2.8  27±10  ND  ND  H83D/H86D  3.1±1.9  26±8  ND  ND  PfFNR  NADPH-dependent cyt c reduction   NADPH-dependent diaphorase assay   Km for PfFd (μM)  kcat (s−1)  Km for NADPH (μM)  kcat (s−1)  Wild-type  1.3±0.5  25±1  110±40  59±6  R98E  11±3  19±4  74±21  81±17  H157D  9.5±4.4  10±1  59±11  56±11  H286D  0.96±0.70  2.9±0.3  760±450  20±11  K287E  7.9±1.9  21±2  ND  ND  R290E  8.9±5.2  14±4  ND  ND  K298E  8.2±2.2  32±7  ND  ND  K308E  15±6  22±5  ND  ND  K309E  6.2±3.5  15±3  67±12  88±20  K73E/H75D  6.0±2.8  27±10  ND  ND  H83D/H86D  3.1±1.9  26±8  ND  ND  The kcat values are expressed as the numbers per electron equivalent. The values are mean ± SD of at least three independent measurements. ‘ND’ stands for ‘Not determined’. As for the effect of mutations near the NADPH binding site, the diaphorase activity of H286D mutant at the same NADPH concentration for the cyt c reduction assay (500 μM) was considerably low (9.5 s−1), while the activities of K287E and R290E mutants (53 and 65 s−1, respectively) was comparable to that of wild-type PfFNR (63 s−1). Physical binding analysis of Pf FNR mutants for Pf Fd Fd-binding ability of the PfFNR mutants was analysed by pull-down assay using PfFd-immobilized resin (Fig. 3). The resulting binding strength appeared to be decreased in the order of wild-type, H83D/H86D, K309E, H286D, K73E/H75D, H157D, K298E, R98E, K287E, R290E and K308E PfFNRs. The tendency of the changes in the Fd-binding strength is similar to that of the Km for PfFd obtained by the kinetic analysis described above (summarized as the numbers of * and + in Fig. 1). Fig. 3 View largeDownload slide Pull-down assay of PfFNRs. PfFNR proteins before loading to the resin (lane O) and bound to immobilized PfFd (lane B) were analysed by SDS PAGE. Protein bands were quantified using densitometry (ImageJ), and the binding ability of each PfFNR (%intensity of the bands of lane B relative to those of lane O) was shown as the numbers above each lanes. Partial cleavage products (about 24 kDa) were also included for the analysis. The broad band around 15 kDa observed in the lanes B corresponds to PfFd which is thought to be released from the PfFd-immobilized resin during heating and denaturation processes in the SDS PAGE sample buffer. Most of the ferredoxins show the slower migration than expected from the molecular size based on its amino acid sequence (in case of PfFd; 11 kDa). Fig. 3 View largeDownload slide Pull-down assay of PfFNRs. PfFNR proteins before loading to the resin (lane O) and bound to immobilized PfFd (lane B) were analysed by SDS PAGE. Protein bands were quantified using densitometry (ImageJ), and the binding ability of each PfFNR (%intensity of the bands of lane B relative to those of lane O) was shown as the numbers above each lanes. Partial cleavage products (about 24 kDa) were also included for the analysis. The broad band around 15 kDa observed in the lanes B corresponds to PfFd which is thought to be released from the PfFd-immobilized resin during heating and denaturation processes in the SDS PAGE sample buffer. Most of the ferredoxins show the slower migration than expected from the molecular size based on its amino acid sequence (in case of PfFd; 11 kDa). Thermodynamics of PfFd binding to PfFNRs was analysed by isothermal calorimetry (ITC; Fig. 4 and Table II), using wild-type PfFNR and two PfFNR mutants (R98E and K73E/H75D) which showed the decreased affinity for PfFd in the kinetic and pull-down analyses. The titration of wild-type PfFNR to PfFd showed a series of heat peaks indicating complex formation with heat uptake (Fig. 4), which is similar to the previous results obtained with maize leaf Fd and FNR (29). While the positive ΔHbind value displays energetically unfavourable endothermic binding reaction, negative values of ΔGbind and −TΔSbind indicate spontaneous Fd: FNR complex formation driven by entropy gain (positive ΔSbind; Table II). In the measurement of the two mutant PfFNRs, endothermic binding heat was also detected (Fig. 4). The resulting dissociation constant (Kd) of the two mutants (27.8 and 23.1 μM) is several times higher than that of wild-type PfFNR (3.25 μM, Table II), which is consistent with the results of the kinetic (Table I) and pull-down (Fig. 3) analyses. In comparison to wild-type PfFNR, the complex formation was unfavoured by negative entropy change in the case of R98E, while by positive enthalpy change in the case of K73E/H75D. Table II. Thermodynamic parameters of the complex formation between PfFd and PfFNRs obtained by ITC PfFNR  Kd (μM)  ΔGbind (kcal mol−1)  ΔHbind (kcal mol−1)  −TΔSbind (kcal mol−1)  Wild-type  3.25  −7.5  8.0  −15.5  R98E  27.8  −6.2  7.4  −13.7  K73E/H75D  23.1  −6.3  8.9  −15.2  PfFNR  Kd (μM)  ΔGbind (kcal mol−1)  ΔHbind (kcal mol−1)  −TΔSbind (kcal mol−1)  Wild-type  3.25  −7.5  8.0  −15.5  R98E  27.8  −6.2  7.4  −13.7  K73E/H75D  23.1  −6.3  8.9  −15.2  Table II. Thermodynamic parameters of the complex formation between PfFd and PfFNRs obtained by ITC PfFNR  Kd (μM)  ΔGbind (kcal mol−1)  ΔHbind (kcal mol−1)  −TΔSbind (kcal mol−1)  Wild-type  3.25  −7.5  8.0  −15.5  R98E  27.8  −6.2  7.4  −13.7  K73E/H75D  23.1  −6.3  8.9  −15.2  PfFNR  Kd (μM)  ΔGbind (kcal mol−1)  ΔHbind (kcal mol−1)  −TΔSbind (kcal mol−1)  Wild-type  3.25  −7.5  8.0  −15.5  R98E  27.8  −6.2  7.4  −13.7  K73E/H75D  23.1  −6.3  8.9  −15.2  Fig. 4 View largeDownload slide Thermodynamics of binding reactions between PfFd and PfFNRs using ITC. ITC thermograms of the titration of PfFd to wild-type (left), R98E (middle) and K73E/75D (right) PfFNRs are shown in the upper panels, and normalized heat values were plotted against the molar ratio ([Fd]/[FNR]) in the lower panels. Fig. 4 View largeDownload slide Thermodynamics of binding reactions between PfFd and PfFNRs using ITC. ITC thermograms of the titration of PfFd to wild-type (left), R98E (middle) and K73E/75D (right) PfFNRs are shown in the upper panels, and normalized heat values were plotted against the molar ratio ([Fd]/[FNR]) in the lower panels. Electron transfer rate from Pf FNR mutants to Pf Fd mutants of D26N/E29Q/E34Q, D65N/E66Q and E92Q/D93N/D97N In order to gain insight into the interaction mode between PfFNR and PfFd, electron transfer rate from the PfFNR mutants to PfFd mutants of D26N/E29Q/E34Q, D65N/E66Q and E92Q/D93N/D97N, which were previously shown to affect the PfFNR binding (24), was measured (Table III and Supplementary Fig. S3). Six PfFNR mutants which significantly reduce the electron transfer to PfFd at 5.0 μM were used for the analysis. Wild-type PfFNR showed large (about 60%) reduction in the electron transfer to D26N/E29Q/E34Q PfFd, and moderate (about 25%) reduction to D65N/E66Q PfFd, compared to wild-type PfFd (Table III and Supplementary Fig. S3), consistent with previous results (Km for wild-type, D26N/E29Q/E34Q and D65N/E66Q PfFds were 2.2, 10.3 and 7.2 μM, respectively) (24). The effect of PfFd mutations were varied among the PfFNR mutants examined. H157D PfFNR mutant showed less reduction (about 15%) in the electron transfer to D26N/E29Q/E34Q PfFd, while larger (about 50%) reduction to D65N/E66Q PfFd. On the other hand, K287E and R290E PfFNR mutants showed no reduction in the electron transfer to D65N/E66Q PfFd compared to wild-type PfFd. R98E showed a larger (about 50%) reduction in the electron transfer to D65N/E66Q PfFd, which is similar to H157D, while the other two PfFNR mutants (K298E and K308E) showed large (about 60–70%) reduction in the electron transfer to D26N/E29Q/E34Q PfFd, and moderate (about 20–30%) reduction to D65N/E66Q PfFd, which is similar to wild-type PfFNRs. For the electron transfer from PfFNR to E92Q/D93N/D97N PfFd mutant, previous study showed the slight (1.3 times) increase in Km and 1.6 times increase in kcat, compared to wild-type PfFd (24). In this study, K287E showed a much less increase in the electron transfer rate to this Fd, while K308E showed a larger increase, compared to wild-type PfFd. Other four PfFNR mutants showed a similar increase in the electron transfer rate to this Fd with that of wild-type PfFNR. Table III. NADPH-dependent cyt c reductase activity of wild-type and mutated PfFNRs using wild-type and mutated PfFds PfFNR  NADPH-dependent cyt c reductase activity at 5.0 μM PfFd (s−1) (% activity vs. wt PfFd)   wild-type PfFd  D26N/E29Q/E34Q  D65N/E66Q  E92Q/D93N/D97N  Wild-type  9.5±1.3(100)  4.0±0.9(43)  7.2±1.7(77)  19±5(200)  R98E  5.3±2.4(100)  3.1±0.9 (58)  2.4±1.3(45)  9.6±1.1(180)  H157D  3.9±1.0(100)  3.3±0.8 (86)  1.9±0.6(50)  11±3(290)  K287E  3.1±0.4(100)  1.7±0.4(55)  3.1±0.6(100)  3.9±0.5(130)  R290E  1.5±0.6(100)  0.83±0.00(55)  1.4±0.2(91)  3.5±1.0(230)  K298E  4.0±1.0(100)  1.5±0.2(38)  3.3±0.0(83)  9.6±1.3(240)  K308E  2.2±0.6(100)  0.70±0.64(31)  1.5±0.6(69)  7.8±0.6(350)  PfFNR  NADPH-dependent cyt c reductase activity at 5.0 μM PfFd (s−1) (% activity vs. wt PfFd)   wild-type PfFd  D26N/E29Q/E34Q  D65N/E66Q  E92Q/D93N/D97N  Wild-type  9.5±1.3(100)  4.0±0.9(43)  7.2±1.7(77)  19±5(200)  R98E  5.3±2.4(100)  3.1±0.9 (58)  2.4±1.3(45)  9.6±1.1(180)  H157D  3.9±1.0(100)  3.3±0.8 (86)  1.9±0.6(50)  11±3(290)  K287E  3.1±0.4(100)  1.7±0.4(55)  3.1±0.6(100)  3.9±0.5(130)  R290E  1.5±0.6(100)  0.83±0.00(55)  1.4±0.2(91)  3.5±1.0(230)  K298E  4.0±1.0(100)  1.5±0.2(38)  3.3±0.0(83)  9.6±1.3(240)  K308E  2.2±0.6(100)  0.70±0.64(31)  1.5±0.6(69)  7.8±0.6(350)  The values are mean ± SD of at least three independent measurements. Relative activity (%) compared to that of wild-type PfFd with each PfFNR is shown in parenthesis. Table III. NADPH-dependent cyt c reductase activity of wild-type and mutated PfFNRs using wild-type and mutated PfFds PfFNR  NADPH-dependent cyt c reductase activity at 5.0 μM PfFd (s−1) (% activity vs. wt PfFd)   wild-type PfFd  D26N/E29Q/E34Q  D65N/E66Q  E92Q/D93N/D97N  Wild-type  9.5±1.3(100)  4.0±0.9(43)  7.2±1.7(77)  19±5(200)  R98E  5.3±2.4(100)  3.1±0.9 (58)  2.4±1.3(45)  9.6±1.1(180)  H157D  3.9±1.0(100)  3.3±0.8 (86)  1.9±0.6(50)  11±3(290)  K287E  3.1±0.4(100)  1.7±0.4(55)  3.1±0.6(100)  3.9±0.5(130)  R290E  1.5±0.6(100)  0.83±0.00(55)  1.4±0.2(91)  3.5±1.0(230)  K298E  4.0±1.0(100)  1.5±0.2(38)  3.3±0.0(83)  9.6±1.3(240)  K308E  2.2±0.6(100)  0.70±0.64(31)  1.5±0.6(69)  7.8±0.6(350)  PfFNR  NADPH-dependent cyt c reductase activity at 5.0 μM PfFd (s−1) (% activity vs. wt PfFd)   wild-type PfFd  D26N/E29Q/E34Q  D65N/E66Q  E92Q/D93N/D97N  Wild-type  9.5±1.3(100)  4.0±0.9(43)  7.2±1.7(77)  19±5(200)  R98E  5.3±2.4(100)  3.1±0.9 (58)  2.4±1.3(45)  9.6±1.1(180)  H157D  3.9±1.0(100)  3.3±0.8 (86)  1.9±0.6(50)  11±3(290)  K287E  3.1±0.4(100)  1.7±0.4(55)  3.1±0.6(100)  3.9±0.5(130)  R290E  1.5±0.6(100)  0.83±0.00(55)  1.4±0.2(91)  3.5±1.0(230)  K298E  4.0±1.0(100)  1.5±0.2(38)  3.3±0.0(83)  9.6±1.3(240)  K308E  2.2±0.6(100)  0.70±0.64(31)  1.5±0.6(69)  7.8±0.6(350)  The values are mean ± SD of at least three independent measurements. Relative activity (%) compared to that of wild-type PfFd with each PfFNR is shown in parenthesis. Comparison of electron transfer rate from Pf FNR mutants to Pf Fd and plant Fds PfFNR preferentially interacts with PfFd over plant Fds; Km values for root Fd and leaf Fd from maize are several times and more than 10 times higher than that for PfFd, respectively (7). Whether the PfFNR mutations also affect the electron transfer to these plant Fds was investigated using PfFd and maize root Fd at 5.0 μM and maize leaf Fd at 40 μM (Table IV and Supplementary Fig. S4). General decrease in the activity was observed for the six mutants of R98E, H157D, K287E, R290E, K298E and K308E. Among them, two mutants (K287E and R290E) showed the selective decreases in the activity with PfFd as compared to plant Fds (Supplementary Fig. S4). Table IV. NADPH-dependent cyt c reductase activity of wild-type and mutated PfFNRs using PfFd and plant Fds PfFNR  NADPH-dependent cyt c reductase activity   5.0 μM PfFd (s−1)  5.0 μM root Fd (s−1)  40 μM leaf Fd (s−1)  Wild-type  8.6±0.5  3.6±0.9  2.2±0.2  R98E  4.7±0.2  2.8±0.5  1.1±0.2  H157D  2.6±0.2  1.9±0.5  0.70±0.48  H286D  ND  ND  ND  K287E  1.5±0.5  1.7±0.4  1.0±0.2  R290E  1.5±0.2  1.5±0.2  1.0±0.2  K298E  3.5±1.2  2.5±1.1  1.3±0.0  K308E  2.9±0.0  1.9±0.5  0.83±0.00  K309E  8.7±1.5  3.3±0.7  1.9±0.5  K73E/H75D  9.6±0.4  3.5±0.5  2.1±0.4  H83D/H86D  7.4±0.6  2.9±0.4  1.9±0.2  PfFNR  NADPH-dependent cyt c reductase activity   5.0 μM PfFd (s−1)  5.0 μM root Fd (s−1)  40 μM leaf Fd (s−1)  Wild-type  8.6±0.5  3.6±0.9  2.2±0.2  R98E  4.7±0.2  2.8±0.5  1.1±0.2  H157D  2.6±0.2  1.9±0.5  0.70±0.48  H286D  ND  ND  ND  K287E  1.5±0.5  1.7±0.4  1.0±0.2  R290E  1.5±0.2  1.5±0.2  1.0±0.2  K298E  3.5±1.2  2.5±1.1  1.3±0.0  K308E  2.9±0.0  1.9±0.5  0.83±0.00  K309E  8.7±1.5  3.3±0.7  1.9±0.5  K73E/H75D  9.6±0.4  3.5±0.5  2.1±0.4  H83D/H86D  7.4±0.6  2.9±0.4  1.9±0.2  The values are mean ± SD of at least three independent measurements. ‘ND’ stands for ‘Not determined’. Table IV. NADPH-dependent cyt c reductase activity of wild-type and mutated PfFNRs using PfFd and plant Fds PfFNR  NADPH-dependent cyt c reductase activity   5.0 μM PfFd (s−1)  5.0 μM root Fd (s−1)  40 μM leaf Fd (s−1)  Wild-type  8.6±0.5  3.6±0.9  2.2±0.2  R98E  4.7±0.2  2.8±0.5  1.1±0.2  H157D  2.6±0.2  1.9±0.5  0.70±0.48  H286D  ND  ND  ND  K287E  1.5±0.5  1.7±0.4  1.0±0.2  R290E  1.5±0.2  1.5±0.2  1.0±0.2  K298E  3.5±1.2  2.5±1.1  1.3±0.0  K308E  2.9±0.0  1.9±0.5  0.83±0.00  K309E  8.7±1.5  3.3±0.7  1.9±0.5  K73E/H75D  9.6±0.4  3.5±0.5  2.1±0.4  H83D/H86D  7.4±0.6  2.9±0.4  1.9±0.2  PfFNR  NADPH-dependent cyt c reductase activity   5.0 μM PfFd (s−1)  5.0 μM root Fd (s−1)  40 μM leaf Fd (s−1)  Wild-type  8.6±0.5  3.6±0.9  2.2±0.2  R98E  4.7±0.2  2.8±0.5  1.1±0.2  H157D  2.6±0.2  1.9±0.5  0.70±0.48  H286D  ND  ND  ND  K287E  1.5±0.5  1.7±0.4  1.0±0.2  R290E  1.5±0.2  1.5±0.2  1.0±0.2  K298E  3.5±1.2  2.5±1.1  1.3±0.0  K308E  2.9±0.0  1.9±0.5  0.83±0.00  K309E  8.7±1.5  3.3±0.7  1.9±0.5  K73E/H75D  9.6±0.4  3.5±0.5  2.1±0.4  H83D/H86D  7.4±0.6  2.9±0.4  1.9±0.2  The values are mean ± SD of at least three independent measurements. ‘ND’ stands for ‘Not determined’. Discussion In this study, basic amino acid residues on the FAD-binding surface of PfFNR were shown to be involved in the interaction with PfFd to different extents as depicted in Fig. 1. Results of the electron transfer kinetics and physical binding analyses indicate that the mutations of R98E, H157D, K287E, R290E, K298E and K308E conferred the larger effects, and the mutations of K309E and K73E/H75D to a less extent, on the interaction between PfFNR and PfFd. Among these residues, R98, H157, R290 and K308 are Plasmodium-specific residues which are not conserved among other FNRs, and K73/H75 are located in the Plasumodium-specific insertion region (Supplementary Fig. S1B). Only one residue (K287) is conserved among most FNRs and the rest of two residues (K298 and K309) are conserved among Plasmodium sp. and root-type FNRs. Therefore, the majority of these basic residues are Plasmodium-specific and not conserved among plants and cyanobacteria. The results of electron transfer rate from Pf FNR mutants to D26N/E29Q/E34Q, D65N/E66Q and E92Q/D93N/D97N PfFds led us to propose possible electrostatic interactions between PfFNR and PfFd as follows (depicted in Fig. 5). H157 (possibly R98) of PfFNR may interact with D26/E29/E34 of PfFd because the basic to acidic mutation of these sites on PfFNR moderated the effect of the mutation of D26N/E29Q/E34Q on the electron transfer to PfFd, as compared to wild-type PfFNR, while it conferred larger reduction of electron transfer to D65N/E66Q PfFd mutant (synergetic effect). Similarly, K287 and R290 may interact with D65/E66 of PfFd because K287E and R290E mutations cancelled the effect of D65N/E66Q on the electron transfer to PfFd. K298E and K308E mutants showed similar pattern to wild-type PfFNR, which suggests that K298 and K308 may interact with the region other than D26/E29/E34 and D65/E66 of PfFd. For the electron transfer from PfFNR to E92Q/D93N/D97N PfFd mutant, the affinity for the Fd was previously shown to slightly decrease, but kcat increased possibly because of the increase in the electron transfer efficiency such as the changes in the redox potential of PfFd (24). K308E PfFNR mutant significantly increased the electron transfer to this E92Q/D93N/D97N PfFd mutant (vs. PfFd) as compared to wild-type PfFNR, possibly because K308 interacts with E92/D93/D97 (C-terminal region) of PfFd. Based on these consideration, we propose a possible interaction mode of PfFNR and PfFd, as shown in Fig. 5, which is almost opposite to that of leaf Fd-FNR complex and intermediate of root and Anabaena complexes, in terms of the orientation of Fd relative to FNR. Fig. 5 View largeDownload slide Structures and possible interaction of PfFNR and PfFd. PfFNR with mutated basic residues are shown as described in the legend of Fig. 1, and PfFd with possible FNR-interacting residues are shown as sticks on the surface of the crystal structures (PDB code 1IUE for PfFd). Broad arrows stand for the possible interaction sites between PfFd and PfFNR proposed in this study. Fig. 5 View largeDownload slide Structures and possible interaction of PfFNR and PfFd. PfFNR with mutated basic residues are shown as described in the legend of Fig. 1, and PfFd with possible FNR-interacting residues are shown as sticks on the surface of the crystal structures (PDB code 1IUE for PfFd). Broad arrows stand for the possible interaction sites between PfFd and PfFNR proposed in this study. PfFNR preferentially interacts with PfFd over root and leaf Fds with several times and more than 10 times differences in the Km values, respectively (7). Therefore, the Fd interaction sites on PfFNR could be categorized into the general Fd binding sites, Plasmodium Fd-specific binding sites and/or non-photosynthetic-type (root and Plasmodium) Fd binding sites. Whether the basic residues found in this study could be classified into these groups were investigated (Table IV and Supplementary Fig. S4). K287E and R290E (and possibly K298E) mutations appeared to selectively decrease the electron transfer to PfFd, suggesting that these residues involve the Plasmodium-Fd specific interaction. D26/E29/E34, D65/E66 and E92/D93/D97 of PfFd residues examined above are mostly conserved among Fd species, except for D97 at the C-terminus which is specific to Plasmodium Fd. There are three clusters of Plasmodium Fd-specific residues at R30/Q31/N32, N58/D59 and C-terminus (H96/D97/M98) surrounding its [Fe-S] cluster (Supplementary Fig. S1A). Considering the proposed orientation depicted in Fig. 5, K287 and K290 may interact also with N58/D59, and K298 may interact also with the C-terminus region, and thus confer the Plasmodium Fd-selective interaction; amino acid residues at the 58/59 positions for leaf and root Fds are Q/S, and at the C-terminus are Y for root Fd and T/G/A for leaf Fd. Further analyses of the structure and interaction between PfFd and PfFNR should clarify the detailed interaction modes (such as salt-bridges and hydrogen bonds) and the relative orientation of the two proteins. The interaction of Fd and FNR appears to be optimized through differentiation of their function in the plastids, depending on the direction of electron transfer and/or the nature of Fd-dependent enzymes. Apicoplasts in malaria parasites contain the essential metabolic pathways unique to Plasmodium sp. in order to survive within the cells of insects or animal hosts at various stages. This study suggests that the interaction of PfFd: PfFNR is diverged so that it meets the physiological requirement in the cells of Plasmodium species. Supplementary Data Supplementary Data are available at JB Online. Funding This work was supported by Grants-in-Aids for Scientific Research (C) [23570165 to Y.K.] from the Japan Society for the Promotion of Science. Conflict of Interest None declared. References 1 McFadden G.I., Reith M.E., Munholland J., Lang-Unnasch N. ( 1996) Plastid in human parasites. Nature  381, 482 Google Scholar CrossRef Search ADS PubMed  2 Köhler S., Delwiche C.F., Denny P.W., Tilney L.G., Webster P., Wilson R.J., Palmer J.D., Roos D.S. ( 1997) A plastid of probable green algal origin in apicomplexan parasites. Science  275, 1485– 14891997) Google Scholar CrossRef Search ADS PubMed  3 Fichera M.E., Roos D.S. ( 1997) A plastid organelle as a drug target in apicomplexan parasites. Nature  390, 407– 409 Google Scholar CrossRef Search ADS PubMed  4 McFadden G.I., Roos D.S. ( 1999) Apicomplexan plastids as drug targets. Trends Microbiol.  7, 328– 333 Google Scholar CrossRef Search ADS PubMed  5 Yeh E., DeRisi J.L. ( 2011) Chemical rescue of malaria parasites lacking an apicoplast defines organelle function in blood-stage. Plasmodium falciparum. PLoS Biol . 9, e100138 Google Scholar CrossRef Search ADS   6 Goodman C.D., McFadden G.I. ( 2013) Targeting apicoplasts in malaria parasites. Expert Opin. Ther. Targets  17, 167– 177 Google Scholar CrossRef Search ADS PubMed  7 Kimata-Ariga Y., Kurisu G., Kusunoki M., Aoki S., Sato D., Kobayashi T., Kita K., Horii T., Hase T. ( 2006) Cloning and characterization of ferredoxin and ferredoxin-NADP+ reductase from human malaria parasite. J. Biochem . 141, 421– 428 Google Scholar CrossRef Search ADS   8 Knaff D.B. ( 1996) Ferredoxin and ferredoxin-dependent enzymes in oxygenic photosynthesis: the light reactions in Advances in Photosynthesis and Respiration  ( Ort D.R., Yocum C.F., eds.) Vol. 4, pp. 333–361, Kluwer Academic Publishers, Springer, Dordrecht 9 Hanke G., Mulo P. ( 2013) Plant type ferredoxins and ferredoxin-dependent metabolism. Plant Cell Environ . 36, 1071– 1084 Google Scholar CrossRef Search ADS PubMed  10 Hanke G., Goss T. ( 2014) The end of the line: can ferredoxin and ferredoxin NADP(H) oxidoreductase determine the fate of photosynthetic electrons? Curr. Protein Pept. Sci . 15, 385– 393 Google Scholar CrossRef Search ADS PubMed  11 Bowsher C.G., Hucklesby D.P., Emes M.J. ( 1993) Induction of ferredoxin-NADP+ oxidoreductase and ferredoxin synthesis in pea root plastids during nitrate assimilation. Plant J . 3, 463– 467 Google Scholar CrossRef Search ADS   12 Emes M.J., Neuhaus H.E. ( 1997) Metabolism and transport in non-photosynthetic plastids. J. Exp. Bot.  48, 1995– 2005 13 Seeber F. ( 2002) Biogenesis of iron-sulphur clusters in amitochondriate and apicomplexan protists. Int. J. Parasitol.  32, 1207– 1217 Google Scholar CrossRef Search ADS PubMed  14 Rohrich R.C., Englert N., Troschke K., Reichenberg A., Hintz M., Seeber F., Balconi E., Aliverti A., Zanetti G., Kohler U., Pfeiffer M., Beck E., Jomaa H., Wiesner J. ( 2005) Reconstitution of an apicoplast-localized electron transfer pathway involved in the isoprenoid biosynthesis of Plasmodium falciparum. FEBS Lett . 579, 6433– 6438 Google Scholar CrossRef Search ADS PubMed  15 Saggu G.S., Garg S., Pala Z.R., Yadav S.K., Kochar S.K., Kochar D.K., Saxena V. ( 2017) Characterization of 4-hydroxy-3-methylbut-2-en-1-yl diphosphate synthase (IspG) from Plasmodium vivax and its potential as an antimalarial drug target. Int. J. Biol. Macromol . 96, 466– 473 Google Scholar CrossRef Search ADS PubMed  16 Vollmer M., Thomsen N., Wiek S., Seeber F. ( 2001) Apicomplexan parasites possess distinct nuclear-encoded, but apicoplast-localized, plant-type ferredoxin-NADP+ reductase and ferredoxin. J. Biol. Chem.  276, 5483– 5490 Google Scholar CrossRef Search ADS PubMed  17 Okada K. ( 2009) The novel heme oxygenase-like protein from Plasmodium falciparum converts heme to bilirubin IXa in the apicoplast. FEBS Lett . 583, 313– 319 Google Scholar CrossRef Search ADS PubMed  18 Suwito H., Jumina M., Pudjiastuti P., Fanani M.Z., Kimata-Ariga Y., Katahira R., Kawakami T., Fujiwara T., Hase T., Sirat H.M., Puspaningsih N.N. ( 2014) Design and synthesis of chalcone derivatives as inhibitor of ferredoxin-ferredoxin-NADP+ reductase interaction of Plasmodium falciparum: pursuing new antimalarial agents. Molecules  19, 21473– 21488 Google Scholar CrossRef Search ADS PubMed  19 Pandini V.H., Caprini G., Thomsen N., Aliverti A., Seeber F., Zanetti G. ( 2002) Ferredoxin-NADP+ reductase and ferredoxin of the protozoan parasite Toxoplasma gondii interact productively in vitro and in vivo. J. Biol. Chem.  277, 48463– 48471 Google Scholar CrossRef Search ADS PubMed  20 Bednarek A., Wiek S., Lingelbach K., Seeber F. ( 2003) Toxoplasma gondii: analysis of the active site insertion of its ferredoxin-NADP+ reductase by peptide-specific antibodies and homology-based modeling. Exp. Parasitol . 103, 68– 77 Google Scholar CrossRef Search ADS PubMed  21 Milani M., Balconi E., Aliverti A., Mastrangelo E., Seeber F., Bolognesi M., Zanetti G. ( 2007) Ferredoxin-NADP+ reductase from Plasmodium falciparum undergoes NADP+-dependent dimerization and inactivation: functional and crystallographic analysis. J. Mol. Biol . 367, 501– 513 Google Scholar CrossRef Search ADS PubMed  22 Balconi E., Pennati A., Crobu D. ( 2009) The ferredoxin-NADP+ reductase/ferredoxin electron transfer system of Plasmodium falciparum. FEBS J . 276, 3825– 3836 Google Scholar CrossRef Search ADS PubMed  23 Crobu D., Canevari G., Milani M. ( 2009) Plasmodium falciparum ferredoxin-NADP+ reductase His286 plays a dual role in NADP(H) binding and catalysis. Biochemistry  48, 9525– 9533 Google Scholar CrossRef Search ADS PubMed  24 Kimata-Ariga Y., Saitoh T., Ikegami T., Horii T., Hase T. ( 2007) Molecular interaction of ferredoxin and ferredoxin-NADP+ reductase from human malaria parasite. J. Biochem . 142, 715– 720 Google Scholar CrossRef Search ADS PubMed  25 Kurisu G., Kusunoki M., Katoh E., Yamazaki T., Teshima K., Onda Y., Kimata-Ariga Y., Hase T. ( 2001) Structure of the electron transfer complex between ferredoxin and ferredoxin-NADP+ reductase. Nat. Struct. Biol.  8, 117– 121 Google Scholar CrossRef Search ADS PubMed  26 Shinohara F., Kurisu G., Hase T., Kimata-Ariga Y. ( 2017) Structural basis for the isotype-specific interactions of ferredoxin and ferredoxin: NADP+ oxidoreductase: an evolutionary switch between photosynthetic and heterotrophic assimilation. Photosynth. Res . 134, 281– 289 Google Scholar CrossRef Search ADS PubMed  27 Morales R., Charon M.H., Kachalova G., Serre L., Medina M., Gómez-Moreno C., Frey M. ( 2000) A redox-dependent interaction between two electron-transfer partners involved in photosynthesis. EMBO Rep . 1, 271– 276 Google Scholar CrossRef Search ADS PubMed  28 Onda Y., Matsumura T., Kimata-Ariga Y., Sakakibara H., Sugiyama T., Hase T. ( 2000) Differential interaction of maize root ferredoxin: NADP+ oxidoreductase with photosynthetic and non-photosynthetic ferredoxin isoproteins. Plant Physiol . 123, 1037– 1045 Google Scholar CrossRef Search ADS PubMed  29 Kinoshita M., Kim J.Y., Kume S., Sakakibara Y., Sugiki T., Kojima C., Kurisu G., Ikegami T., Hase T., Kimata-Ariga Y., Lee Y.H. ( 2015) Physicochemical nature of interfaces controlling ferredoxin NADP+ reductase activity through its interprotein interactions with ferredoxin. Biochim. Biophys. Acta  1847, 1200– 1211 Google Scholar CrossRef Search ADS PubMed  Abbreviations Abbreviations cyt c cytochrome c Fd ferredoxin FNR ferredoxin-NADP+reductase ITC isothermal titration calorimetry PfFd Plasmodium falciparum ferredoxin PfFNR Plasmodium falciparum ferredoxin-NADP+reductase © The Author(s) 2018. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png The Journal of Biochemistry Oxford University Press

Plasmodium-specific basic amino acid residues important for the interaction with ferredoxin on the surface of ferredoxin-NADP+ reductase

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Oxford University Press
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© The Author(s) 2018. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved
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0021-924X
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1756-2651
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Abstract

Abstract The malaria parasite (Plasmodium falciparum) possesses a plastid-derived, essential organelle called the apicoplast, which contains a redox system comprising plant-type ferredoxin (Fd) and Fd-NADP+ reductase (FNR). This system supplies reducing power for the crucial metabolic pathways in this organelle. Electron transfer between P. falciparum Fd (PfFd) and FNR (PfFNR) is performed with higher affinity and specificity than that of plant Fd and FNR. To investigate the mechanism for such superior protein–protein interaction, we searched for the Fd interaction sites on the surface of PfFNR. Basic amino acid residues on the FAD binding side of PfFNR were comprehensively substituted to acidic amino acids by site-directed mutagenesis. Kinetic analysis of electron transfer to PfFd and plant Fds, physical binding to immobilized PfFd and thermodynamics of the PfFd binding using these PfFNR mutants revealed that several basic amino acid residues including those in Plasmodium-specific insertion region are important for the interaction with PfFd. Majority of these basic residues are Plasmodium-specific and not conserved among plant and cyanobacteria FNRs. These results suggest that the interaction mode of Fd and FNR is diverged during evolution so that PfFd: PfFNR interaction meets the physiological requirement in the cells of Plasmodium species. ferredoxin, ferredoxin-NADP+, reductase, malaria parasite, plastid evolution, protein–protein interaction Protozoan parasites of the phylum Apicomplexa, including Plasmodium sp. (the causative agent of malaria) and Toxoplasma sp., contain a non-photosynthetic plastid organelle called the apicoplast (1), which was acquired by secondary endosymbiosis of algae (2). The apicoplast was shown to be essential for the parasite’s survival (3, 4) and therefore considered as an ideal drug target without distressing the animal host. The vital function of the apicoplast has been investigated; the biosynthesis of isoprenoids was shown to be the only essential role in the erythrocytic life stage (5), while the biosynthesis of fatty acids, Fe-S clusters and hemes may be essential in other stages such as the liver stage or the mosquito stage (6). We previously reported the cloning and characterization of plant-type ferredoxin (Fd) and Fd-NADP+ reductase (FNR) from Plasmodium falciparum (human malaria parasite), and proposed that they constitute a redox system which supplies reducing power that drives the biosynthetic reactions in the apicoplast (7). In higher plants, Fd is mainly reduced by the photosynthetic electron transport chain and donates reducing equivalents to FNR and other Fd-dependent enzymes involved in various metabolic and regulatory reactions in the plastid (8–10). In non-photosynthetic tissues such as roots, these Fd-dependent enzymes are also functioning, but the reduction of Fd is catalysed by FNR, using NADPH generated through the oxidative pentose phosphate cycle (11). These discrete redox cascades are conducted by the combination of genetically distinct isoforms of Fd and FNR present differentially in photosynthetic and non-photosynthetic tissues (12). By analogy to the non-photosynthetic type in plants, a redox cascade of NADPH–FNR–Fd has been considered to provide reducing power for the crucial Fd-dependent metabolisms in the apicoplasts (5). The involvement of apicoplast Fd has been indicated in the reactions of an Fe-S cluster assembly pathway (13), isoprenoid biosynthesis pathway (14, 15), fatty acid desaturation (16) and heme oxygenation (17). Because this plant-type redox system is not present in the mammalian host, it would represent a promising drug target to combat malaria, for instance, by developing substances which inhibit the interaction between Fd and FNR from malaria parasites (18). The amino acid sequence of P. falciparum Fd (PfFd) (Supplementary Fig. S1A) shares about 50% homology with plant Fds (16, 19), and its backbone structure, as solved by X-ray crystallography, closely resembles to those of plant Fds (7). On the other hand, P. falciparum FNR (PfFNR) (Supplementary Fig. S1B) shares lower homology (20–30%) with plant FNRs, displaying large insertions and deletions specific to Plasmodium sp. (20) which appear to be responsible for the unique disordered surface structures shown by its crystal structure analysis (21). In a reconstituted assay system, electron transfer between PfFd and PfFNR is performed with higher affinity for the Fd (about 1 μM of Km) (7) than that of plant Fd: FNR combination (3–4 μM of Km). In addition, PfFNR transfers electrons preferentially to PfFd with much higher (several to 10 times) affinity over plant Fds (7), indicating their advantageous interaction. Since crystal structures of PfFd (7) and PfFNR (21) have been determined, several studies have addressed the structure–function characteristics of PfFNR (21–23), but the molecular interaction between PfFd and PfFNR has not been addressed well, except for our previous study (24). Fd and FNR form 1:1 complex for the efficient electron transfer between their prosthetic groups of the 2Fe-2S cluster and FAD, respectively. X-ray crystal structures of the complexes of Fd and FNR from leaf (25) and root (26) in higher plants and from cyanobacteria (Anabaena PCC7119) (27) have been reported, clarifying the sites involved in the complex formation between the two protein molecules. Unexpectedly, the interaction mode of Fd and FNR, such as the binding surface area and the orientation of Fd relative to FNR, is largely different between the Fd–FNR pairs of photosynthetic (leaf) type and non-photosynthetic (root) type (26). These distinct Fd–FNR interaction modes between the two types of the complexes may be due to the optimization for their efficiency in photosynthetic and non-photosynthetic (heterotrophic) electron transfer cascades, which are opposite in terms of the direction of electron flow (26) as described above. The Fd-FNR interaction mode of the Anabaena complex which physiologically operates both directions of electron flow is also different from either of the plant complexes (27). However, there is a common feature that multiple salt bridges mostly between acidic residues of Fd and basic residues of FNR stabilize the complex formation; there are five salt bridges in the leaf complex, three in the root complex and two in the Anabaena complex. The amino acid residues involved in these salt bridges are mostly conserved among Fd species, but largely different among FNRs. Thus, FNR interacts with Fd using the amino acid residues specific to each FNR species, which determines the iso-type specific orientation of Fd relative to FNR (26). So far, the 3D structure of the PfFd–PfFNR complex is not available, but several acidic amino acid residues on PfFd (e.g. D26, E29, E34, D65 and E66) were shown to be important for the interaction with PfFNR by the NMR chemical shift perturbation analysis combined with site-directed mutagenesis to neutral amino acids (24). Therefore, the electrostatic interaction between these acidic residues in PfFd and certain basic residues in PfFNR is expected to be important for the complex formation. In this study, we found that several basic amino acid residues on the FAD binding side of PfFNR are important for the interaction with Pf Fd, and that majority of these FNR residues are specific to Plasmodium sp. and not conserved among plants and cyanobacteria. Materials and Methods Site-directed mutagenesis of PfFNR and preparation of recombinant proteins Cloning and preparation of PfFNR, PfFd (wild-type and mutants) and maize Fds were described previously (7, 24, 28). For the construction of PfFNR mutants, the QuikChange site-directed mutagenesis kit (Agilent Technologies, USA) was used according to the manufacturer’s instructions. The synthetic oligonucleotides used for the mutagenesis are shown in Supplementary Table S1. The mutation sites and the sequence integrity of the entire coding region of PfFNR were confirmed by DNA sequencing. Enzymatic analysis Enzyme activity of PfFNR was measured using a grating microplate reader (model SH-1000 Lab, CORONA, Japan). The activity of NADPH-dependent electron transfer from FNR to Fd was measured using cytochrome c (cyt c) as a final electron acceptor as described previously (28) except that the concentrations of PfFNR and NADPH were increased to 50 nM and 500 μM, respectively. Diaphorase activity of FNR with DCPIP as an electron acceptor was measured as described previously (28) except that the concentration of PfFNR was lowered to 25 nM and NaCl at 100 mM was included. Pull-down assay Immobilized PfFd was obtained by coupling PfFd to CNBr-activated-Sepharose 4B (GE Healthcare Bio-Science, USA), according to the manufacturer’s directions. PfFNR mutants (0.20 nmol) were incubated with 20 μl of the immobilized PfFd resin (containing 4.5 nmol of PfFd) in the solution of 50 mM Tris–HCl, pH 7.5, 100 mM NaCl, and the resin was washed three times with the same solution and centrifuged. The resulting resin pellet was extracted with 50 μl of SDS sample buffer, and 10 μl of the extract was analysed by 12.5% SDS PAGE. Isothermal titration calorimetry (ITC) Protein samples were dialyzed against 50 mM Tris–HCl, pH 7.5, 100 mM NaCl, and degassed for 3 min before being loaded into the calorimeter. Calorimetric experiments were performed with an Auto-iTC200 instrument (GE Healthcare Biosciences) at 298 K. In the injection syringe, 500 μM PfFd was titrated into 50 μM wild-type or mutant PfFNR in the ITC cell. Titration experiments consisted of 38 injections spaced at intervals of 150 s. The injection volume was 1.0 μl and the cell was continuously stirred at 1000 rpm. Thermodynamic parameters of the complex formation between Fd and FNR were obtained as described previously (29). Results Preparation of site-directed mutants of Pf FNR Basic amino acid residues on the molecular surface of the FAD binding side of PfFNR (Fig. 1) were comprehensively mutated to acidic residues. Eleven mutants were prepared; two double mutants (K73E/H75D and H83D/H86D) and one triple mutant (H93D/K95E/K96E) in the Plasmodium-specific insertion region (shown as dotted line in Fig. 1), and eight single mutants (R98E, H157D, H286D, K287E, R290E, K298E, K308E and K309E) in other regions. UV-visible spectra of these 11 mutants showed no significant difference from that of wild-type PfFNR (7), indicating that the holo-form enzymes containing FAD were produced. SDS PAGE analysis of the mutant PfFNRs (Fig. 2) showed similar mobility on the gel except that K73E/H75D mutant showed slightly slower mobility and H93D/K95E/K96E mutant appeared to be mostly cleaved during storage and/or during SDS PAGE (full-size FNR protein fragment was observed in the fresh preparation of this mutant by MALDI-TOF mass analysis as shown in Supplementary Fig. S2). Therefore, PfFNR mutants other than the triple mutant were used for the further analyses. Fig. 1 View largeDownload slide Molecular surface of PfFNR with basic residues mutated in this study. Mutated basic residues are shown as sticks with mesh, and FAD and NADPH-analogue, adenosine 2′, 5′-diphosphate (A2P), are shown as sticks on the X-ray crystal structure of PfFNR (PDB code 2OK7). There are five disordered surface regions in the structure which could not be presented as surface model, and among them, the region from N61 to I94 in a Plasmodium-specific long insertion is shown as dotted line. The number of * and + depicted beside the residue numbers stand for the extent of the changes in the Km value and the physical Fd-binding, respectively, analysed in this study. ND stands for ‘not determined’. Fig. 1 View largeDownload slide Molecular surface of PfFNR with basic residues mutated in this study. Mutated basic residues are shown as sticks with mesh, and FAD and NADPH-analogue, adenosine 2′, 5′-diphosphate (A2P), are shown as sticks on the X-ray crystal structure of PfFNR (PDB code 2OK7). There are five disordered surface regions in the structure which could not be presented as surface model, and among them, the region from N61 to I94 in a Plasmodium-specific long insertion is shown as dotted line. The number of * and + depicted beside the residue numbers stand for the extent of the changes in the Km value and the physical Fd-binding, respectively, analysed in this study. ND stands for ‘not determined’. Fig. 2 View largeDownload slide SDS PAGE analysis of purified Pf FNRs. Wild-type and mutant PfFNR proteins (about 1 μg each) were analysed by 12.5% SDS PAGE and stained with coomassie brilliant blue. Triple mutant of H93D/K95E/K96E was mostly cleaved as detailed in the text. Partially cleaved products (20–24 kDa) were also observed with other FNRs. Fig. 2 View largeDownload slide SDS PAGE analysis of purified Pf FNRs. Wild-type and mutant PfFNR proteins (about 1 μg each) were analysed by 12.5% SDS PAGE and stained with coomassie brilliant blue. Triple mutant of H93D/K95E/K96E was mostly cleaved as detailed in the text. Partially cleaved products (20–24 kDa) were also observed with other FNRs. Kinetic analysis of electron transfer from Pf FNR mutants to Pf Fd The activity of NADPH-dependent electron transfer from the 10 PfFNR mutants to PfFd was measured (Table I). The PfFNR mutants except for H286D and H83D/H86D showed significant increases in the Km for PfFd as compared to wild-type PfFNR, indicating that the charge reversal mutation of only one or two residues in the eight PfFNR mutants considerably affects the interaction with PfFd. The extent of the effect is variable (from 4 to 11 times of Km value) depending on the mutation site, and the average Km value increased in the order of K73E/H75D, K309E, K287E, K298E, R290E, H157D, R98E and K308E PfFNRs. H286D mutant did not show significant change in the Km but exhibited a large decrease in the kcat (2.9 s−1) as compared to other PfFNRs (10–32 s−1) (Table I). H286 is located in the vicinity of the NADPH binding site (A2P, an NADPH analogue, is shown in Fig. 1) and its hydrogen-bonding ability was suggested to be important for the binding of NADPH (23). Measurement of NADPH-dependent diaphorase activity of H286D mutant showed a large decrease in the affinity for NADPH (several times increase in the Km; Table I) as compared to wild-type PfFNR, which mostly explains the observed reduction in the electron transfer (cyt c reduction) activity performed at 500 μM NADPH. For other FNRs analyzed, the Km values for NADPH were much lower than 500 μM NADPH, and the kcat values for the diaphorase activity were more than 2.4 times higher than those of cyt c reduction, indicating that the FNR reduction by NADPH is not a rate-limiting step in the cyt c reduction assay under the experimental conditions in this study. Table I. Steady-state kinetic parameters of wild-type and mutated PfFNRs in the reactions of NADPH-dependent cyt c reduction using PfFd and of NADPH-dependent diaphorase assay using DCPIP PfFNR  NADPH-dependent cyt c reduction   NADPH-dependent diaphorase assay   Km for PfFd (μM)  kcat (s−1)  Km for NADPH (μM)  kcat (s−1)  Wild-type  1.3±0.5  25±1  110±40  59±6  R98E  11±3  19±4  74±21  81±17  H157D  9.5±4.4  10±1  59±11  56±11  H286D  0.96±0.70  2.9±0.3  760±450  20±11  K287E  7.9±1.9  21±2  ND  ND  R290E  8.9±5.2  14±4  ND  ND  K298E  8.2±2.2  32±7  ND  ND  K308E  15±6  22±5  ND  ND  K309E  6.2±3.5  15±3  67±12  88±20  K73E/H75D  6.0±2.8  27±10  ND  ND  H83D/H86D  3.1±1.9  26±8  ND  ND  PfFNR  NADPH-dependent cyt c reduction   NADPH-dependent diaphorase assay   Km for PfFd (μM)  kcat (s−1)  Km for NADPH (μM)  kcat (s−1)  Wild-type  1.3±0.5  25±1  110±40  59±6  R98E  11±3  19±4  74±21  81±17  H157D  9.5±4.4  10±1  59±11  56±11  H286D  0.96±0.70  2.9±0.3  760±450  20±11  K287E  7.9±1.9  21±2  ND  ND  R290E  8.9±5.2  14±4  ND  ND  K298E  8.2±2.2  32±7  ND  ND  K308E  15±6  22±5  ND  ND  K309E  6.2±3.5  15±3  67±12  88±20  K73E/H75D  6.0±2.8  27±10  ND  ND  H83D/H86D  3.1±1.9  26±8  ND  ND  The kcat values are expressed as the numbers per electron equivalent. The values are mean ± SD of at least three independent measurements. ‘ND’ stands for ‘Not determined’. As for the effect of mutations near the NADPH binding site, the diaphorase activity of H286D mutant at the same NADPH concentration for the cyt c reduction assay (500 μM) was considerably low (9.5 s−1), while the activities of K287E and R290E mutants (53 and 65 s−1, respectively) was comparable to that of wild-type PfFNR (63 s−1). Table I. Steady-state kinetic parameters of wild-type and mutated PfFNRs in the reactions of NADPH-dependent cyt c reduction using PfFd and of NADPH-dependent diaphorase assay using DCPIP PfFNR  NADPH-dependent cyt c reduction   NADPH-dependent diaphorase assay   Km for PfFd (μM)  kcat (s−1)  Km for NADPH (μM)  kcat (s−1)  Wild-type  1.3±0.5  25±1  110±40  59±6  R98E  11±3  19±4  74±21  81±17  H157D  9.5±4.4  10±1  59±11  56±11  H286D  0.96±0.70  2.9±0.3  760±450  20±11  K287E  7.9±1.9  21±2  ND  ND  R290E  8.9±5.2  14±4  ND  ND  K298E  8.2±2.2  32±7  ND  ND  K308E  15±6  22±5  ND  ND  K309E  6.2±3.5  15±3  67±12  88±20  K73E/H75D  6.0±2.8  27±10  ND  ND  H83D/H86D  3.1±1.9  26±8  ND  ND  PfFNR  NADPH-dependent cyt c reduction   NADPH-dependent diaphorase assay   Km for PfFd (μM)  kcat (s−1)  Km for NADPH (μM)  kcat (s−1)  Wild-type  1.3±0.5  25±1  110±40  59±6  R98E  11±3  19±4  74±21  81±17  H157D  9.5±4.4  10±1  59±11  56±11  H286D  0.96±0.70  2.9±0.3  760±450  20±11  K287E  7.9±1.9  21±2  ND  ND  R290E  8.9±5.2  14±4  ND  ND  K298E  8.2±2.2  32±7  ND  ND  K308E  15±6  22±5  ND  ND  K309E  6.2±3.5  15±3  67±12  88±20  K73E/H75D  6.0±2.8  27±10  ND  ND  H83D/H86D  3.1±1.9  26±8  ND  ND  The kcat values are expressed as the numbers per electron equivalent. The values are mean ± SD of at least three independent measurements. ‘ND’ stands for ‘Not determined’. As for the effect of mutations near the NADPH binding site, the diaphorase activity of H286D mutant at the same NADPH concentration for the cyt c reduction assay (500 μM) was considerably low (9.5 s−1), while the activities of K287E and R290E mutants (53 and 65 s−1, respectively) was comparable to that of wild-type PfFNR (63 s−1). Physical binding analysis of Pf FNR mutants for Pf Fd Fd-binding ability of the PfFNR mutants was analysed by pull-down assay using PfFd-immobilized resin (Fig. 3). The resulting binding strength appeared to be decreased in the order of wild-type, H83D/H86D, K309E, H286D, K73E/H75D, H157D, K298E, R98E, K287E, R290E and K308E PfFNRs. The tendency of the changes in the Fd-binding strength is similar to that of the Km for PfFd obtained by the kinetic analysis described above (summarized as the numbers of * and + in Fig. 1). Fig. 3 View largeDownload slide Pull-down assay of PfFNRs. PfFNR proteins before loading to the resin (lane O) and bound to immobilized PfFd (lane B) were analysed by SDS PAGE. Protein bands were quantified using densitometry (ImageJ), and the binding ability of each PfFNR (%intensity of the bands of lane B relative to those of lane O) was shown as the numbers above each lanes. Partial cleavage products (about 24 kDa) were also included for the analysis. The broad band around 15 kDa observed in the lanes B corresponds to PfFd which is thought to be released from the PfFd-immobilized resin during heating and denaturation processes in the SDS PAGE sample buffer. Most of the ferredoxins show the slower migration than expected from the molecular size based on its amino acid sequence (in case of PfFd; 11 kDa). Fig. 3 View largeDownload slide Pull-down assay of PfFNRs. PfFNR proteins before loading to the resin (lane O) and bound to immobilized PfFd (lane B) were analysed by SDS PAGE. Protein bands were quantified using densitometry (ImageJ), and the binding ability of each PfFNR (%intensity of the bands of lane B relative to those of lane O) was shown as the numbers above each lanes. Partial cleavage products (about 24 kDa) were also included for the analysis. The broad band around 15 kDa observed in the lanes B corresponds to PfFd which is thought to be released from the PfFd-immobilized resin during heating and denaturation processes in the SDS PAGE sample buffer. Most of the ferredoxins show the slower migration than expected from the molecular size based on its amino acid sequence (in case of PfFd; 11 kDa). Thermodynamics of PfFd binding to PfFNRs was analysed by isothermal calorimetry (ITC; Fig. 4 and Table II), using wild-type PfFNR and two PfFNR mutants (R98E and K73E/H75D) which showed the decreased affinity for PfFd in the kinetic and pull-down analyses. The titration of wild-type PfFNR to PfFd showed a series of heat peaks indicating complex formation with heat uptake (Fig. 4), which is similar to the previous results obtained with maize leaf Fd and FNR (29). While the positive ΔHbind value displays energetically unfavourable endothermic binding reaction, negative values of ΔGbind and −TΔSbind indicate spontaneous Fd: FNR complex formation driven by entropy gain (positive ΔSbind; Table II). In the measurement of the two mutant PfFNRs, endothermic binding heat was also detected (Fig. 4). The resulting dissociation constant (Kd) of the two mutants (27.8 and 23.1 μM) is several times higher than that of wild-type PfFNR (3.25 μM, Table II), which is consistent with the results of the kinetic (Table I) and pull-down (Fig. 3) analyses. In comparison to wild-type PfFNR, the complex formation was unfavoured by negative entropy change in the case of R98E, while by positive enthalpy change in the case of K73E/H75D. Table II. Thermodynamic parameters of the complex formation between PfFd and PfFNRs obtained by ITC PfFNR  Kd (μM)  ΔGbind (kcal mol−1)  ΔHbind (kcal mol−1)  −TΔSbind (kcal mol−1)  Wild-type  3.25  −7.5  8.0  −15.5  R98E  27.8  −6.2  7.4  −13.7  K73E/H75D  23.1  −6.3  8.9  −15.2  PfFNR  Kd (μM)  ΔGbind (kcal mol−1)  ΔHbind (kcal mol−1)  −TΔSbind (kcal mol−1)  Wild-type  3.25  −7.5  8.0  −15.5  R98E  27.8  −6.2  7.4  −13.7  K73E/H75D  23.1  −6.3  8.9  −15.2  Table II. Thermodynamic parameters of the complex formation between PfFd and PfFNRs obtained by ITC PfFNR  Kd (μM)  ΔGbind (kcal mol−1)  ΔHbind (kcal mol−1)  −TΔSbind (kcal mol−1)  Wild-type  3.25  −7.5  8.0  −15.5  R98E  27.8  −6.2  7.4  −13.7  K73E/H75D  23.1  −6.3  8.9  −15.2  PfFNR  Kd (μM)  ΔGbind (kcal mol−1)  ΔHbind (kcal mol−1)  −TΔSbind (kcal mol−1)  Wild-type  3.25  −7.5  8.0  −15.5  R98E  27.8  −6.2  7.4  −13.7  K73E/H75D  23.1  −6.3  8.9  −15.2  Fig. 4 View largeDownload slide Thermodynamics of binding reactions between PfFd and PfFNRs using ITC. ITC thermograms of the titration of PfFd to wild-type (left), R98E (middle) and K73E/75D (right) PfFNRs are shown in the upper panels, and normalized heat values were plotted against the molar ratio ([Fd]/[FNR]) in the lower panels. Fig. 4 View largeDownload slide Thermodynamics of binding reactions between PfFd and PfFNRs using ITC. ITC thermograms of the titration of PfFd to wild-type (left), R98E (middle) and K73E/75D (right) PfFNRs are shown in the upper panels, and normalized heat values were plotted against the molar ratio ([Fd]/[FNR]) in the lower panels. Electron transfer rate from Pf FNR mutants to Pf Fd mutants of D26N/E29Q/E34Q, D65N/E66Q and E92Q/D93N/D97N In order to gain insight into the interaction mode between PfFNR and PfFd, electron transfer rate from the PfFNR mutants to PfFd mutants of D26N/E29Q/E34Q, D65N/E66Q and E92Q/D93N/D97N, which were previously shown to affect the PfFNR binding (24), was measured (Table III and Supplementary Fig. S3). Six PfFNR mutants which significantly reduce the electron transfer to PfFd at 5.0 μM were used for the analysis. Wild-type PfFNR showed large (about 60%) reduction in the electron transfer to D26N/E29Q/E34Q PfFd, and moderate (about 25%) reduction to D65N/E66Q PfFd, compared to wild-type PfFd (Table III and Supplementary Fig. S3), consistent with previous results (Km for wild-type, D26N/E29Q/E34Q and D65N/E66Q PfFds were 2.2, 10.3 and 7.2 μM, respectively) (24). The effect of PfFd mutations were varied among the PfFNR mutants examined. H157D PfFNR mutant showed less reduction (about 15%) in the electron transfer to D26N/E29Q/E34Q PfFd, while larger (about 50%) reduction to D65N/E66Q PfFd. On the other hand, K287E and R290E PfFNR mutants showed no reduction in the electron transfer to D65N/E66Q PfFd compared to wild-type PfFd. R98E showed a larger (about 50%) reduction in the electron transfer to D65N/E66Q PfFd, which is similar to H157D, while the other two PfFNR mutants (K298E and K308E) showed large (about 60–70%) reduction in the electron transfer to D26N/E29Q/E34Q PfFd, and moderate (about 20–30%) reduction to D65N/E66Q PfFd, which is similar to wild-type PfFNRs. For the electron transfer from PfFNR to E92Q/D93N/D97N PfFd mutant, previous study showed the slight (1.3 times) increase in Km and 1.6 times increase in kcat, compared to wild-type PfFd (24). In this study, K287E showed a much less increase in the electron transfer rate to this Fd, while K308E showed a larger increase, compared to wild-type PfFd. Other four PfFNR mutants showed a similar increase in the electron transfer rate to this Fd with that of wild-type PfFNR. Table III. NADPH-dependent cyt c reductase activity of wild-type and mutated PfFNRs using wild-type and mutated PfFds PfFNR  NADPH-dependent cyt c reductase activity at 5.0 μM PfFd (s−1) (% activity vs. wt PfFd)   wild-type PfFd  D26N/E29Q/E34Q  D65N/E66Q  E92Q/D93N/D97N  Wild-type  9.5±1.3(100)  4.0±0.9(43)  7.2±1.7(77)  19±5(200)  R98E  5.3±2.4(100)  3.1±0.9 (58)  2.4±1.3(45)  9.6±1.1(180)  H157D  3.9±1.0(100)  3.3±0.8 (86)  1.9±0.6(50)  11±3(290)  K287E  3.1±0.4(100)  1.7±0.4(55)  3.1±0.6(100)  3.9±0.5(130)  R290E  1.5±0.6(100)  0.83±0.00(55)  1.4±0.2(91)  3.5±1.0(230)  K298E  4.0±1.0(100)  1.5±0.2(38)  3.3±0.0(83)  9.6±1.3(240)  K308E  2.2±0.6(100)  0.70±0.64(31)  1.5±0.6(69)  7.8±0.6(350)  PfFNR  NADPH-dependent cyt c reductase activity at 5.0 μM PfFd (s−1) (% activity vs. wt PfFd)   wild-type PfFd  D26N/E29Q/E34Q  D65N/E66Q  E92Q/D93N/D97N  Wild-type  9.5±1.3(100)  4.0±0.9(43)  7.2±1.7(77)  19±5(200)  R98E  5.3±2.4(100)  3.1±0.9 (58)  2.4±1.3(45)  9.6±1.1(180)  H157D  3.9±1.0(100)  3.3±0.8 (86)  1.9±0.6(50)  11±3(290)  K287E  3.1±0.4(100)  1.7±0.4(55)  3.1±0.6(100)  3.9±0.5(130)  R290E  1.5±0.6(100)  0.83±0.00(55)  1.4±0.2(91)  3.5±1.0(230)  K298E  4.0±1.0(100)  1.5±0.2(38)  3.3±0.0(83)  9.6±1.3(240)  K308E  2.2±0.6(100)  0.70±0.64(31)  1.5±0.6(69)  7.8±0.6(350)  The values are mean ± SD of at least three independent measurements. Relative activity (%) compared to that of wild-type PfFd with each PfFNR is shown in parenthesis. Table III. NADPH-dependent cyt c reductase activity of wild-type and mutated PfFNRs using wild-type and mutated PfFds PfFNR  NADPH-dependent cyt c reductase activity at 5.0 μM PfFd (s−1) (% activity vs. wt PfFd)   wild-type PfFd  D26N/E29Q/E34Q  D65N/E66Q  E92Q/D93N/D97N  Wild-type  9.5±1.3(100)  4.0±0.9(43)  7.2±1.7(77)  19±5(200)  R98E  5.3±2.4(100)  3.1±0.9 (58)  2.4±1.3(45)  9.6±1.1(180)  H157D  3.9±1.0(100)  3.3±0.8 (86)  1.9±0.6(50)  11±3(290)  K287E  3.1±0.4(100)  1.7±0.4(55)  3.1±0.6(100)  3.9±0.5(130)  R290E  1.5±0.6(100)  0.83±0.00(55)  1.4±0.2(91)  3.5±1.0(230)  K298E  4.0±1.0(100)  1.5±0.2(38)  3.3±0.0(83)  9.6±1.3(240)  K308E  2.2±0.6(100)  0.70±0.64(31)  1.5±0.6(69)  7.8±0.6(350)  PfFNR  NADPH-dependent cyt c reductase activity at 5.0 μM PfFd (s−1) (% activity vs. wt PfFd)   wild-type PfFd  D26N/E29Q/E34Q  D65N/E66Q  E92Q/D93N/D97N  Wild-type  9.5±1.3(100)  4.0±0.9(43)  7.2±1.7(77)  19±5(200)  R98E  5.3±2.4(100)  3.1±0.9 (58)  2.4±1.3(45)  9.6±1.1(180)  H157D  3.9±1.0(100)  3.3±0.8 (86)  1.9±0.6(50)  11±3(290)  K287E  3.1±0.4(100)  1.7±0.4(55)  3.1±0.6(100)  3.9±0.5(130)  R290E  1.5±0.6(100)  0.83±0.00(55)  1.4±0.2(91)  3.5±1.0(230)  K298E  4.0±1.0(100)  1.5±0.2(38)  3.3±0.0(83)  9.6±1.3(240)  K308E  2.2±0.6(100)  0.70±0.64(31)  1.5±0.6(69)  7.8±0.6(350)  The values are mean ± SD of at least three independent measurements. Relative activity (%) compared to that of wild-type PfFd with each PfFNR is shown in parenthesis. Comparison of electron transfer rate from Pf FNR mutants to Pf Fd and plant Fds PfFNR preferentially interacts with PfFd over plant Fds; Km values for root Fd and leaf Fd from maize are several times and more than 10 times higher than that for PfFd, respectively (7). Whether the PfFNR mutations also affect the electron transfer to these plant Fds was investigated using PfFd and maize root Fd at 5.0 μM and maize leaf Fd at 40 μM (Table IV and Supplementary Fig. S4). General decrease in the activity was observed for the six mutants of R98E, H157D, K287E, R290E, K298E and K308E. Among them, two mutants (K287E and R290E) showed the selective decreases in the activity with PfFd as compared to plant Fds (Supplementary Fig. S4). Table IV. NADPH-dependent cyt c reductase activity of wild-type and mutated PfFNRs using PfFd and plant Fds PfFNR  NADPH-dependent cyt c reductase activity   5.0 μM PfFd (s−1)  5.0 μM root Fd (s−1)  40 μM leaf Fd (s−1)  Wild-type  8.6±0.5  3.6±0.9  2.2±0.2  R98E  4.7±0.2  2.8±0.5  1.1±0.2  H157D  2.6±0.2  1.9±0.5  0.70±0.48  H286D  ND  ND  ND  K287E  1.5±0.5  1.7±0.4  1.0±0.2  R290E  1.5±0.2  1.5±0.2  1.0±0.2  K298E  3.5±1.2  2.5±1.1  1.3±0.0  K308E  2.9±0.0  1.9±0.5  0.83±0.00  K309E  8.7±1.5  3.3±0.7  1.9±0.5  K73E/H75D  9.6±0.4  3.5±0.5  2.1±0.4  H83D/H86D  7.4±0.6  2.9±0.4  1.9±0.2  PfFNR  NADPH-dependent cyt c reductase activity   5.0 μM PfFd (s−1)  5.0 μM root Fd (s−1)  40 μM leaf Fd (s−1)  Wild-type  8.6±0.5  3.6±0.9  2.2±0.2  R98E  4.7±0.2  2.8±0.5  1.1±0.2  H157D  2.6±0.2  1.9±0.5  0.70±0.48  H286D  ND  ND  ND  K287E  1.5±0.5  1.7±0.4  1.0±0.2  R290E  1.5±0.2  1.5±0.2  1.0±0.2  K298E  3.5±1.2  2.5±1.1  1.3±0.0  K308E  2.9±0.0  1.9±0.5  0.83±0.00  K309E  8.7±1.5  3.3±0.7  1.9±0.5  K73E/H75D  9.6±0.4  3.5±0.5  2.1±0.4  H83D/H86D  7.4±0.6  2.9±0.4  1.9±0.2  The values are mean ± SD of at least three independent measurements. ‘ND’ stands for ‘Not determined’. Table IV. NADPH-dependent cyt c reductase activity of wild-type and mutated PfFNRs using PfFd and plant Fds PfFNR  NADPH-dependent cyt c reductase activity   5.0 μM PfFd (s−1)  5.0 μM root Fd (s−1)  40 μM leaf Fd (s−1)  Wild-type  8.6±0.5  3.6±0.9  2.2±0.2  R98E  4.7±0.2  2.8±0.5  1.1±0.2  H157D  2.6±0.2  1.9±0.5  0.70±0.48  H286D  ND  ND  ND  K287E  1.5±0.5  1.7±0.4  1.0±0.2  R290E  1.5±0.2  1.5±0.2  1.0±0.2  K298E  3.5±1.2  2.5±1.1  1.3±0.0  K308E  2.9±0.0  1.9±0.5  0.83±0.00  K309E  8.7±1.5  3.3±0.7  1.9±0.5  K73E/H75D  9.6±0.4  3.5±0.5  2.1±0.4  H83D/H86D  7.4±0.6  2.9±0.4  1.9±0.2  PfFNR  NADPH-dependent cyt c reductase activity   5.0 μM PfFd (s−1)  5.0 μM root Fd (s−1)  40 μM leaf Fd (s−1)  Wild-type  8.6±0.5  3.6±0.9  2.2±0.2  R98E  4.7±0.2  2.8±0.5  1.1±0.2  H157D  2.6±0.2  1.9±0.5  0.70±0.48  H286D  ND  ND  ND  K287E  1.5±0.5  1.7±0.4  1.0±0.2  R290E  1.5±0.2  1.5±0.2  1.0±0.2  K298E  3.5±1.2  2.5±1.1  1.3±0.0  K308E  2.9±0.0  1.9±0.5  0.83±0.00  K309E  8.7±1.5  3.3±0.7  1.9±0.5  K73E/H75D  9.6±0.4  3.5±0.5  2.1±0.4  H83D/H86D  7.4±0.6  2.9±0.4  1.9±0.2  The values are mean ± SD of at least three independent measurements. ‘ND’ stands for ‘Not determined’. Discussion In this study, basic amino acid residues on the FAD-binding surface of PfFNR were shown to be involved in the interaction with PfFd to different extents as depicted in Fig. 1. Results of the electron transfer kinetics and physical binding analyses indicate that the mutations of R98E, H157D, K287E, R290E, K298E and K308E conferred the larger effects, and the mutations of K309E and K73E/H75D to a less extent, on the interaction between PfFNR and PfFd. Among these residues, R98, H157, R290 and K308 are Plasmodium-specific residues which are not conserved among other FNRs, and K73/H75 are located in the Plasumodium-specific insertion region (Supplementary Fig. S1B). Only one residue (K287) is conserved among most FNRs and the rest of two residues (K298 and K309) are conserved among Plasmodium sp. and root-type FNRs. Therefore, the majority of these basic residues are Plasmodium-specific and not conserved among plants and cyanobacteria. The results of electron transfer rate from Pf FNR mutants to D26N/E29Q/E34Q, D65N/E66Q and E92Q/D93N/D97N PfFds led us to propose possible electrostatic interactions between PfFNR and PfFd as follows (depicted in Fig. 5). H157 (possibly R98) of PfFNR may interact with D26/E29/E34 of PfFd because the basic to acidic mutation of these sites on PfFNR moderated the effect of the mutation of D26N/E29Q/E34Q on the electron transfer to PfFd, as compared to wild-type PfFNR, while it conferred larger reduction of electron transfer to D65N/E66Q PfFd mutant (synergetic effect). Similarly, K287 and R290 may interact with D65/E66 of PfFd because K287E and R290E mutations cancelled the effect of D65N/E66Q on the electron transfer to PfFd. K298E and K308E mutants showed similar pattern to wild-type PfFNR, which suggests that K298 and K308 may interact with the region other than D26/E29/E34 and D65/E66 of PfFd. For the electron transfer from PfFNR to E92Q/D93N/D97N PfFd mutant, the affinity for the Fd was previously shown to slightly decrease, but kcat increased possibly because of the increase in the electron transfer efficiency such as the changes in the redox potential of PfFd (24). K308E PfFNR mutant significantly increased the electron transfer to this E92Q/D93N/D97N PfFd mutant (vs. PfFd) as compared to wild-type PfFNR, possibly because K308 interacts with E92/D93/D97 (C-terminal region) of PfFd. Based on these consideration, we propose a possible interaction mode of PfFNR and PfFd, as shown in Fig. 5, which is almost opposite to that of leaf Fd-FNR complex and intermediate of root and Anabaena complexes, in terms of the orientation of Fd relative to FNR. Fig. 5 View largeDownload slide Structures and possible interaction of PfFNR and PfFd. PfFNR with mutated basic residues are shown as described in the legend of Fig. 1, and PfFd with possible FNR-interacting residues are shown as sticks on the surface of the crystal structures (PDB code 1IUE for PfFd). Broad arrows stand for the possible interaction sites between PfFd and PfFNR proposed in this study. Fig. 5 View largeDownload slide Structures and possible interaction of PfFNR and PfFd. PfFNR with mutated basic residues are shown as described in the legend of Fig. 1, and PfFd with possible FNR-interacting residues are shown as sticks on the surface of the crystal structures (PDB code 1IUE for PfFd). Broad arrows stand for the possible interaction sites between PfFd and PfFNR proposed in this study. PfFNR preferentially interacts with PfFd over root and leaf Fds with several times and more than 10 times differences in the Km values, respectively (7). Therefore, the Fd interaction sites on PfFNR could be categorized into the general Fd binding sites, Plasmodium Fd-specific binding sites and/or non-photosynthetic-type (root and Plasmodium) Fd binding sites. Whether the basic residues found in this study could be classified into these groups were investigated (Table IV and Supplementary Fig. S4). K287E and R290E (and possibly K298E) mutations appeared to selectively decrease the electron transfer to PfFd, suggesting that these residues involve the Plasmodium-Fd specific interaction. D26/E29/E34, D65/E66 and E92/D93/D97 of PfFd residues examined above are mostly conserved among Fd species, except for D97 at the C-terminus which is specific to Plasmodium Fd. There are three clusters of Plasmodium Fd-specific residues at R30/Q31/N32, N58/D59 and C-terminus (H96/D97/M98) surrounding its [Fe-S] cluster (Supplementary Fig. S1A). Considering the proposed orientation depicted in Fig. 5, K287 and K290 may interact also with N58/D59, and K298 may interact also with the C-terminus region, and thus confer the Plasmodium Fd-selective interaction; amino acid residues at the 58/59 positions for leaf and root Fds are Q/S, and at the C-terminus are Y for root Fd and T/G/A for leaf Fd. Further analyses of the structure and interaction between PfFd and PfFNR should clarify the detailed interaction modes (such as salt-bridges and hydrogen bonds) and the relative orientation of the two proteins. The interaction of Fd and FNR appears to be optimized through differentiation of their function in the plastids, depending on the direction of electron transfer and/or the nature of Fd-dependent enzymes. Apicoplasts in malaria parasites contain the essential metabolic pathways unique to Plasmodium sp. in order to survive within the cells of insects or animal hosts at various stages. This study suggests that the interaction of PfFd: PfFNR is diverged so that it meets the physiological requirement in the cells of Plasmodium species. Supplementary Data Supplementary Data are available at JB Online. Funding This work was supported by Grants-in-Aids for Scientific Research (C) [23570165 to Y.K.] from the Japan Society for the Promotion of Science. Conflict of Interest None declared. References 1 McFadden G.I., Reith M.E., Munholland J., Lang-Unnasch N. ( 1996) Plastid in human parasites. Nature  381, 482 Google Scholar CrossRef Search ADS PubMed  2 Köhler S., Delwiche C.F., Denny P.W., Tilney L.G., Webster P., Wilson R.J., Palmer J.D., Roos D.S. ( 1997) A plastid of probable green algal origin in apicomplexan parasites. Science  275, 1485– 14891997) Google Scholar CrossRef Search ADS PubMed  3 Fichera M.E., Roos D.S. ( 1997) A plastid organelle as a drug target in apicomplexan parasites. Nature  390, 407– 409 Google Scholar CrossRef Search ADS PubMed  4 McFadden G.I., Roos D.S. ( 1999) Apicomplexan plastids as drug targets. Trends Microbiol.  7, 328– 333 Google Scholar CrossRef Search ADS PubMed  5 Yeh E., DeRisi J.L. ( 2011) Chemical rescue of malaria parasites lacking an apicoplast defines organelle function in blood-stage. Plasmodium falciparum. PLoS Biol . 9, e100138 Google Scholar CrossRef Search ADS   6 Goodman C.D., McFadden G.I. ( 2013) Targeting apicoplasts in malaria parasites. Expert Opin. Ther. Targets  17, 167– 177 Google Scholar CrossRef Search ADS PubMed  7 Kimata-Ariga Y., Kurisu G., Kusunoki M., Aoki S., Sato D., Kobayashi T., Kita K., Horii T., Hase T. ( 2006) Cloning and characterization of ferredoxin and ferredoxin-NADP+ reductase from human malaria parasite. J. Biochem . 141, 421– 428 Google Scholar CrossRef Search ADS   8 Knaff D.B. ( 1996) Ferredoxin and ferredoxin-dependent enzymes in oxygenic photosynthesis: the light reactions in Advances in Photosynthesis and Respiration  ( Ort D.R., Yocum C.F., eds.) Vol. 4, pp. 333–361, Kluwer Academic Publishers, Springer, Dordrecht 9 Hanke G., Mulo P. ( 2013) Plant type ferredoxins and ferredoxin-dependent metabolism. Plant Cell Environ . 36, 1071– 1084 Google Scholar CrossRef Search ADS PubMed  10 Hanke G., Goss T. ( 2014) The end of the line: can ferredoxin and ferredoxin NADP(H) oxidoreductase determine the fate of photosynthetic electrons? Curr. Protein Pept. Sci . 15, 385– 393 Google Scholar CrossRef Search ADS PubMed  11 Bowsher C.G., Hucklesby D.P., Emes M.J. ( 1993) Induction of ferredoxin-NADP+ oxidoreductase and ferredoxin synthesis in pea root plastids during nitrate assimilation. Plant J . 3, 463– 467 Google Scholar CrossRef Search ADS   12 Emes M.J., Neuhaus H.E. ( 1997) Metabolism and transport in non-photosynthetic plastids. J. Exp. Bot.  48, 1995– 2005 13 Seeber F. ( 2002) Biogenesis of iron-sulphur clusters in amitochondriate and apicomplexan protists. Int. J. Parasitol.  32, 1207– 1217 Google Scholar CrossRef Search ADS PubMed  14 Rohrich R.C., Englert N., Troschke K., Reichenberg A., Hintz M., Seeber F., Balconi E., Aliverti A., Zanetti G., Kohler U., Pfeiffer M., Beck E., Jomaa H., Wiesner J. ( 2005) Reconstitution of an apicoplast-localized electron transfer pathway involved in the isoprenoid biosynthesis of Plasmodium falciparum. FEBS Lett . 579, 6433– 6438 Google Scholar CrossRef Search ADS PubMed  15 Saggu G.S., Garg S., Pala Z.R., Yadav S.K., Kochar S.K., Kochar D.K., Saxena V. ( 2017) Characterization of 4-hydroxy-3-methylbut-2-en-1-yl diphosphate synthase (IspG) from Plasmodium vivax and its potential as an antimalarial drug target. Int. J. Biol. Macromol . 96, 466– 473 Google Scholar CrossRef Search ADS PubMed  16 Vollmer M., Thomsen N., Wiek S., Seeber F. ( 2001) Apicomplexan parasites possess distinct nuclear-encoded, but apicoplast-localized, plant-type ferredoxin-NADP+ reductase and ferredoxin. J. Biol. Chem.  276, 5483– 5490 Google Scholar CrossRef Search ADS PubMed  17 Okada K. ( 2009) The novel heme oxygenase-like protein from Plasmodium falciparum converts heme to bilirubin IXa in the apicoplast. FEBS Lett . 583, 313– 319 Google Scholar CrossRef Search ADS PubMed  18 Suwito H., Jumina M., Pudjiastuti P., Fanani M.Z., Kimata-Ariga Y., Katahira R., Kawakami T., Fujiwara T., Hase T., Sirat H.M., Puspaningsih N.N. ( 2014) Design and synthesis of chalcone derivatives as inhibitor of ferredoxin-ferredoxin-NADP+ reductase interaction of Plasmodium falciparum: pursuing new antimalarial agents. Molecules  19, 21473– 21488 Google Scholar CrossRef Search ADS PubMed  19 Pandini V.H., Caprini G., Thomsen N., Aliverti A., Seeber F., Zanetti G. ( 2002) Ferredoxin-NADP+ reductase and ferredoxin of the protozoan parasite Toxoplasma gondii interact productively in vitro and in vivo. J. Biol. Chem.  277, 48463– 48471 Google Scholar CrossRef Search ADS PubMed  20 Bednarek A., Wiek S., Lingelbach K., Seeber F. ( 2003) Toxoplasma gondii: analysis of the active site insertion of its ferredoxin-NADP+ reductase by peptide-specific antibodies and homology-based modeling. Exp. Parasitol . 103, 68– 77 Google Scholar CrossRef Search ADS PubMed  21 Milani M., Balconi E., Aliverti A., Mastrangelo E., Seeber F., Bolognesi M., Zanetti G. ( 2007) Ferredoxin-NADP+ reductase from Plasmodium falciparum undergoes NADP+-dependent dimerization and inactivation: functional and crystallographic analysis. J. Mol. Biol . 367, 501– 513 Google Scholar CrossRef Search ADS PubMed  22 Balconi E., Pennati A., Crobu D. ( 2009) The ferredoxin-NADP+ reductase/ferredoxin electron transfer system of Plasmodium falciparum. FEBS J . 276, 3825– 3836 Google Scholar CrossRef Search ADS PubMed  23 Crobu D., Canevari G., Milani M. ( 2009) Plasmodium falciparum ferredoxin-NADP+ reductase His286 plays a dual role in NADP(H) binding and catalysis. Biochemistry  48, 9525– 9533 Google Scholar CrossRef Search ADS PubMed  24 Kimata-Ariga Y., Saitoh T., Ikegami T., Horii T., Hase T. ( 2007) Molecular interaction of ferredoxin and ferredoxin-NADP+ reductase from human malaria parasite. J. Biochem . 142, 715– 720 Google Scholar CrossRef Search ADS PubMed  25 Kurisu G., Kusunoki M., Katoh E., Yamazaki T., Teshima K., Onda Y., Kimata-Ariga Y., Hase T. ( 2001) Structure of the electron transfer complex between ferredoxin and ferredoxin-NADP+ reductase. Nat. Struct. Biol.  8, 117– 121 Google Scholar CrossRef Search ADS PubMed  26 Shinohara F., Kurisu G., Hase T., Kimata-Ariga Y. ( 2017) Structural basis for the isotype-specific interactions of ferredoxin and ferredoxin: NADP+ oxidoreductase: an evolutionary switch between photosynthetic and heterotrophic assimilation. Photosynth. Res . 134, 281– 289 Google Scholar CrossRef Search ADS PubMed  27 Morales R., Charon M.H., Kachalova G., Serre L., Medina M., Gómez-Moreno C., Frey M. ( 2000) A redox-dependent interaction between two electron-transfer partners involved in photosynthesis. EMBO Rep . 1, 271– 276 Google Scholar CrossRef Search ADS PubMed  28 Onda Y., Matsumura T., Kimata-Ariga Y., Sakakibara H., Sugiyama T., Hase T. ( 2000) Differential interaction of maize root ferredoxin: NADP+ oxidoreductase with photosynthetic and non-photosynthetic ferredoxin isoproteins. Plant Physiol . 123, 1037– 1045 Google Scholar CrossRef Search ADS PubMed  29 Kinoshita M., Kim J.Y., Kume S., Sakakibara Y., Sugiki T., Kojima C., Kurisu G., Ikegami T., Hase T., Kimata-Ariga Y., Lee Y.H. ( 2015) Physicochemical nature of interfaces controlling ferredoxin NADP+ reductase activity through its interprotein interactions with ferredoxin. Biochim. Biophys. Acta  1847, 1200– 1211 Google Scholar CrossRef Search ADS PubMed  Abbreviations Abbreviations cyt c cytochrome c Fd ferredoxin FNR ferredoxin-NADP+reductase ITC isothermal titration calorimetry PfFd Plasmodium falciparum ferredoxin PfFNR Plasmodium falciparum ferredoxin-NADP+reductase © The Author(s) 2018. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices)

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The Journal of BiochemistryOxford University Press

Published: Apr 23, 2018

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