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Originally published In Press as doi:10.1074/jbc.M408904200 on October 28, 2004

J. Biol. Chem., Vol. 280, Issue 3, 2275-2281, January 21, 2005
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A Structural Basis of Equisetum arvense Ferredoxin Isoform II Producing an Alternative Electron Transfer with Ferredoxin-NADP+ Reductase*

Genji Kurisu{ddagger}§, Daisuke Nishiyama{ddagger}, Masami Kusunoki{ddagger}, Shinobu Fujikawa¶, Midori Katoh¶, Guy Thomas Hanke||, Toshiharu Hase||, and Keizo Teshima¶**

From the {ddagger}Research Center for Structural and Functional Proteomics, Institute for Protein Research, Osaka University, Suita, Osaka 565-0871, Faculty of Integrated Arts and Sciences, Hiroshima University, 1-7-1 Kagamiyama, Higashi-Hiroshima 739-8521, and ||Division of Enzymology, Institute for Protein Research, Osaka University, Suita, Osaka 565-0871, Japan

Received for publication, August 4, 2004 , and in revised form, October 13, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
We have determined the crystal structure, at 1.2-Å resolution, of Equisetum arvense ferredoxin isoform II (FdII), which lacks residues equivalent to Arg39 and Glu28 highly conserved among other ferredoxins (Fds). In other Fds these residues form an intramolecular salt bridge crucial for stabilization of the [2Fe-2S] cluster, which is disrupted upon complex formation with Fd-NADP+ oxidoreductase (FNR) to form two intermolecular salt bridges. The overall structure of FdII resembles the known backbone structures of E. arvense isoform I (FdI) and other plant-type Fds. Dramatically, in the FdII structure a unique, alternative salt bridge is formed between Arg22 and Glu58. This results in a different relative orientation of the {alpha}-helix formed by Leu23-Glu29 and eliminates the possibility of forming three of the five intermolecular salt bridges identified on formation of a complex between maize FdI and maize FNR. Mutation of FdII, informed by structural differences with FdI, showed that the alternative salt bridge and the absence of an otherwise conserved Tyr residue are important for the alternative stabilization of the FdII [2Fe-2S] cluster. We also investigated FdI and FdII electron transfer to FNR on chloroplast thylakoid membranes. The Km and Vmax values of FdII are similar to those of FdI, contrary to previous measurements of the reverse reaction, from FNR to Fd. The affinity between reduced FdI and oxidized FNR is much greater than that between oxidized FdI and reduced FNR, whereas this is not the case with FdII. The pH dependence of electron transfer by FdI, FdII, and an FdII mutant with FdI features was measured and further indicated that the binding mode to FNR differs between FdI and FdII. Based on this evidence, we hypothesize that binding modes with other Fd-dependent reductases may also vary between FdI and FdII. The structural differences between FdI and FdII therefore result in functional differences that may influence partitioning of electrons into different redox metabolic pathways.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
[2Fe-2S] ferredoxin (Fd)1 electron transfer proteins distribute reducing equivalents derived from light energy to Fd-dependent reductases, such as Fd-NADP+ reductase (FNR), nitrite reductase (NiR), sulfite reductase (SiR), and Fd-thioredoxin reductase (FTR), for the assimilation of inorganic compounds and the regulation of carbon assimilation (1, 2). Recently, Fds have also been found to donate electrons to desaturases for biosynthesis of unsaturated fatty acids (3), choline monooxygenase for betaine biosynthesis (4), and heme oxygenase for phytochrome biosynthesis (5), indicative of the contribution of Fds to various metabolic pathways in addition to assimilation of inorganic substances. Most intriguingly, higher plant species possess several isoforms of Fd, implying isoform specificity to different redox-metabolic pathways. For example, maize Fd isoforms, FdI and FdII, are distributed differentially in mesophyll and bundle-sheath cells and are found to be mainly involved in the formation of NADPH and ATP, respectively (6). The functional difference between maize FdI and FdII is dependent solely on the replacement of Asp65 with Asn, which affects the FNR-binding site (7). This variation results in distinct phenotypes of cyanobacteria (lacking an endogenous Fd gene) in which FdI and FdII are expressed (8). Additionally, biochemical experiments indicate partial differences between the Fd-binding sites of the Fd-dependent reductases FNR, SiR, and FTR (9, 10). We therefore suspect that Fd isoforms might play an important role in the modulation of various redox-metabolic pathways in plant cells and that such modulation might be ascribed to specific noncovalent interactions between Fd isoforms and Fd-dependent partner proteins.

Recently, we determined the three-dimensional structure of the maize mesophyll Fd (FdI)·FNR complex at 2.59-Å resolution (11), in which five intermolecular salt bridges are formed between Fd and FNR. This structure shows an intramolecular Fd salt bridge between Arg40-Glu29 is dynamically exchanged to form two intermolecular salt bridges with FNR residues in the process of the complex formation, suggesting the Arg-Glu pair has a role in electron transfer. Furthermore, this Arg-Glu pair is conserved among all but three of more than 70 plant-type Fd sequences (12). In Equisetum arvense the Arg-Glu pair is conserved in Fd isoform I (FdI) but missing from isoform II (FdII), and a comparison of electron transfer with FNR under excess NADPH shows obvious differences between the Km and kcat values of FdI and FdII. Electron transfer rates of FdII mutants, in which the Arg-Glu pair is introduced, indicate that these residues are crucial to these differences (13). These findings suggest that E. arvense FdII might vary from FdI in its electron transfer activity around photosystem I, because of its different interaction with FNR. When the Arg-Glu pair of E. arvense FdI is substituted with the corresponding noncharged residues of FdII (R39Q/E28S), the [2Fe-2S] cluster became unstable (13), indicating a crucial role for these residues in stabilizing Fd in the native state. However, it should be noted that the [2Fe-2S] cluster of wild type FdII is stable even in the absence of a salt bridge between Arg39 and Glu28.

To understand the structural basis of the unique properties of E. arvense FdII and to elucidate the mechanism that stabilizes the [2Fe-2S] cluster, we have therefore determined its crystal structure at 1.2 Å resolution and compared this with the previously determined structure of E. arvense FdI (1.8 Å resolution) (14). We report that FdII has another different internal salt bridge between Arg22-Glu58 instead of that between Arg39-Glu28 and that the [2Fe-2S] cluster is stabilized by this alternative salt bridge, and by the deletion of one amino acid residue in a short loop connecting the cluster binding loop to the preceding {alpha}-helix region. Furthermore, we investigated the electron transfer capabilities of E. arvense FdI and FdII from photosystem I to FNR by using chloroplast thylakoid membranes. We will discuss a correlation between the structural differences of FdI and FdII and their electron transfer abilities with FNR.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Preparation of Recombinant Fds—E. arvense recombinant FdII, FdI, and the FdII mutants were expressed and accumulated as the apo-form in Escherichia coli cells and successfully converted to the holo-form by chemical reconstitution of the [2Fe-2S] cluster as described previously (13). Further purification of the Fds was carried out as described previously (15, 16). Site-specific and insertion mutants of FdII R22T, FdII with Tyr32 newly inserted (FdII(Y32)) and FdII(Y32)Q38R/S28E were prepared with the Quikchange site-directed mutagenesis kit (Stratagene) (13), and these mutation sites were confirmed by DNA sequencing. The concentration of Fd was determined spectrophotometrically based on a molar extinction coefficient of 9.68 mM-1 cm-1 at 422 nm (17).

Crystallization—We searched extensively for crystallization conditions of E. arvense FdII by the hanging drop vapor diffusion method with Crystal Screen I and II (Hampton Research) and Wizard I, II, and cryo (Emerald Biostructures). Sodium phosphate and ammonium sulfate were also checked as precipitants. Needle-shape crystals of FdII were initially obtained at 4 °C from equal volumes of the protein (10 mg/ml) and reservoir solution (3.1 M NaH2PO4/K2HPO4 (pH 7.5)), but were too small for x-ray experiments. Flat plate-shaped crystals of appropriate size were successfully obtained with the same reservoir solution additionally containing benzamidine hydrochloride (2.0% (w/v)). The improved crystals were transferred to a reservoir solution containing sucrose (20% (w/v)) and immediately frozen with liquid nitrogen for x-ray experiments.

Crystallographic Data Collection and Processing—X-ray data were collected at a wavelength of 0.9 Å at beamline BL44XU at the SPring-8 synchrotron in Hyogo, Japan. Diffraction images were collected at liquid nitrogen temperature (100 K) on a CCD-based PX210 detector system. Images were processed with the program DPS/MOSFLM (18) and SCALA in the CCP4 program package (19). Crystal data and crystallographic statistics are given in Table I.


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  		TABLE I
Crystal data, data collection, and refinement statistics

 
Structural Determination and Refinement—The structure was solved by molecular replacement method using E. arvense FdI structure (14) as a search model with the program CNS (20). First, the structure of FdII was refined with isotropic temperature factors using the program CNS. After convergence with CNS, we next used the program SHELXL in the SHELX97 program package to model atomic anisotropy. Manual revision of the atomic model was carried out with the program O (21). On careful inspection of the |Fo| - |Fc| and 2|Fo| - |Fc| maps, we added benzamidine and water molecules into the model and modeled dual conformers of amino acid side chains. Subsequent refinement was carried out up to 1.2 Å resolution without any structural restraints on the [2Fe-2S] cluster. The stereochemical geometry of the model was checked with the program PROCHECK (22).

Stability Assays—The degradation rates of the [2Fe-2S] clusters of FdII and FdII mutants were measured by monitoring the decrease of A420 due to the presence of the [2Fe-2S] cluster for 36 h at pH 7.5, 100 mM NaCl, and 4 °C. The initial values of A420 were set at 0.5-1.2 for all the samples. The stabilities of their [2Fe-2S] clusters were evaluated by their half-lives estimated on the assumption that degradation of the [2Fe-2S] cluster obeys first-order kinetics.

Enzyme Assays—NADP+ photoreduction ability of Fd by purified thylakoid membranes from spinach chloroplast was assayed as described previously (23). The reaction mixture contained, in a total volume of 1.0 ml, 0.2 mM NADP+, thylakoid membranes (about 0.01 µg of chlorophyll), 1-10 µM Fd, 50 mM Hepes (pH 6.4-8.1) and 100 mM NaCl. The reaction was measured in a spectrometer and was initiated by irradiating with red light perpendicular to the spectrophotometric light beam. The increase of A340 due to the photoreduction of NADP+ was monitored. The pH dependences of the abilities of FdI, FdII, and FdII mutant between pH 6.4 and 7.7 were analyzed using the equation, v = V0 + {Sigma}{Delta}Vi x Ka,i/((H+) + Ka,i), where V0 is the ability when all the ionizable groups participating are protonated, and Ka,i is the dissociation constant of the corresponding ionizable group participating, and {Delta}Vi is the change in the ability when the ionizable group with a pKa value of pKa,i is deprotonated. The analysis was not corrected with the effect of a change of the net charge, because only a few ionizable groups are dissociated in the neutral pH region.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Quality of E. arvense FdII Structure—Crystals of FdII belong to space group C2, with unit cell parameters a = 97.54 Å, b = 29.41 Å, c = 32.56 Å, and {beta} = 103.94° (Table I). The refined model of FdII contains 1 FdII molecule, 6 benzamidines, and 208 water molecules in the asymmetric unit. A total of nine residues in the final model have dual conforming side chains. The final crystallographic R factor and free R factor with anisotropic temperature factors are 11.75 and 15.45%, respectively, for 27,366 unique reflections in the resolution range of 16-1.2 Å. All amino acid residues of FdII except glycine and proline residues are in the stereochemically most favored regions in the Ramachandran plot. Refinement statistics are summarized in Table I.

Overall Structure—The overall structures of E. arvense FdII and FdI (14) are shown as ribbon drawings in Fig. 1, A and B, respectively. A superimposition of the main chain structures of FdII and FdI is shown in Fig. 1C. The structures are superimposed along the C-{alpha} atoms of residues 1-93 and the [2Fe-2S] cluster in FdII, and C-{alpha} atoms of residues 1-31 and 33-94 and the [2Fe-2S] cluster in FdI, because FdII lacks the 32nd and the C-terminal 95th residues in comparison to FdI, as shown in the sequence alignment (Fig. 2). The root mean square deviation value for the C-{alpha} atom between the two structures is 1.17 Å. The overall structure of FdII was fairly similar to that of FdI and also resembled the other known backbone structures of plant-type Fds (25, 29-31).



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  		FIG. 1.
General structures and comparison of E. arvense FdII and FdI (14). A, ribbon representation of E. arvense FdII structure with a salt bridge between Arg22-Glu58. The figure is drawn by PyMol (24), and the [2Fe-2S] cluster is represented by magenta and orange balls and stick models. B, ribbon representation of FdI with a salt bridge between Arg39-Glu28. Arrows indicate {alpha}-helices in FdII and FdI with differentially arranged orientations. C, stereo view of C-{alpha} traces of E. arvense FdII and FdI and spinach FdI. FdII is colored in green, FdI in blue, and spinach FdI (25) in red.

 



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  		FIG. 2.
Sequence alignment of E. arvense FdII, FdI (26), maize FdI (27), and spinach FdI (28). Residues different between E. arvense FdII and FdI and conserved among the four Fds are colored red and blue, respectively. Boxed residues represent those that are observed to form an intramolecular salt bridge in either E. arvense FdI or FdII. Residues with filled circles correspond to the residues that are involved in intermolecular salt bridges between maize FdI and FNR (11).

 
However, it should be noted that the backbone conformation connecting the {alpha}-helix of Leu23-Glu29 and cluster binding loop of Pro34-Leu46 in FdII is significantly different from the corresponding conformation of FdI and also that the orientation of {alpha}-helix of Leu23-Glu29 for FdII is different from that of the corresponding {alpha}-helix for FdI by an angle of 27.6°. These differences may be ascribed to the deletion of Tyr32 in FdII (Fig. 2).

In the region of FdII corresponding to the salt bridge formed between Arg39 and Glu28 in FdI (Fig. 1B), the corresponding Gln38 and Ser28 side chains do not even form a hydrogen bond. Instead, Arg22 and Glu58 form a unique, alternative salt bridge in FdII (Fig. 1A), which has no counterpart in the corresponding Thr22 and Glu59 of FdI.

On refinement of this region, we found that both Arg22 and Glu58 have two rotational conformations. In the major side chain conformation, the major electron densities were interpreted as forming a salt bridge, whereas the minor conformations were exposed to the bulk solvent region containing 1.6 M phosphate as a precipitant. Therefore, we consider that these rotational conformations corresponding to minor electron densities were generated because of the very high ionic strength of the surrounding environment and that under the usual physiological conditions the Arg22 and Glu58 form an intramolecular salt bridge.

Structure of the [2Fe-2S] Cluster and Its Surroundings— Inter-atomic distances and bond angles for the [2Fe-2S] cluster in FdII are summarized in Tables II and III, respectively, and also compared with the data for E. arvense FdI (14). These values are quite similar between FdII and FdI.


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  		TABLE II
Inter-atomic distances and their average values for the [2Fe-2S] clusters in FdII and FdI(14) Residue numbers and atom names for FdII are given on the left column and those for FdI in parentheses.

 


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  		TABLE III
Bond angles and their average values for the [2Fe-2S] clusters in FdII and FdI(14) Residue numbers and atom names for FdII are listed on the left column and those for FdI in parentheses.

 
Fe and S atoms of the cluster and C-{alpha} atoms of the cluster binding loop (Pro34-Leu46 for FdII and Pro35-Leu47 for FdI) of FdII and FdI are superimposed with a root mean square deviation value of 0.21 Å. The general arrangement of the [2Fe-2S] cluster and its surroundings show high structural homology between the FdI and FdII. Apart from Leu35, Gln38, and Thr44; the local side chain conformation of the cluster binding loop in FdII is almost identical to that of the corresponding side chains in FdI.

The hydrogen bonding networks around the [2Fe-2S] clusters of FdII and FdI are shown in Fig. 3. In FdII the O-{gamma} atom of Thr44 is hydrogen-bonded to the S-{gamma} atom of Cys42, and an equivalent bond also occurs in FdI between the O-{gamma} atom of Ser45 and the S-{gamma} atom of Cys43. By contrast, FdII has no counterpart to the hydrogen bond between the O-{gamma} atom of Ser37 and the S-{gamma} atom of Cys43 in FdI, because both dual conformations of the side chain of Ser36 for FdII face the bulk solvent region. Therefore, the number of hydrogen bonds between the hydroxyl group of Ser (or Thr) or the backbone amide group and the S atom of the [2Fe-2S] cluster or the sulfhydryl group of the Cys coordinating the cluster is 1 less for FdII than for FdI. In general, it is known that hydrogen bonds between backbone amide groups and cluster S atoms or coordinating sulfhydryl groups decrease the redox potential (32, 33). We also determined the redox potentials of these Fds by cyclic voltammetry demonstrating that the potential (-403.8 ± 0.4 mV) of FdII is slightly lower than that (-396.6 ± 1.3 mV) of FdI. As exposure of the cluster to the bulk solvent and the dielectric constant around the [2Fe-2S] cluster are also crucial factors in determining the redox potential, we are currently refining E. arvense FdI structure at higher resolution (~1.2 Å higher than the known 1.8 Å) to compare more precisely the microenvironment around the [2Fe-2S] cluster at atomic resolution between FdII and FdI. We will report in detail on the relationship between the redox potential and the microenvironment around the cluster, together with the redox potential measurements, elsewhere.



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  		FIG. 3.
Stereo views of the [2Fe-2S] cluster and its surroundings for E. arvense FdII (A) and FdI (B). Hydrogen bonds are indicated by dotted lines.

 
[2Fe-2S] Cluster Stability of FdII and Its Mutants—We reported previously that an E. arvense FdI mutant, substituted at Arg39 and Glu28 with the corresponding noncharged residues of FdII, was unstable and unfolded, releasing its [2Fe-2S] cluster within 15 h (13), whereas the [2Fe-2S] cluster of FdII was stable despite the absence of these salt bridge-forming residues. The present x-ray analysis data show that the conformation of the [2Fe-2S] cluster and the cluster binding loop are almost identical between FdI and FdII, although there is 1 less hydrogen bond around the [2Fe-2S] cluster of FdII (Fig. 3). We also prepared FdI R39Q/E28S/F36L and FdI R39Q/E28S/S45T mutants, in which the cluster binding loops are more similar to that of FdII (Fig. 2) than FdI R39Q/E28S, but these mutations did not restore stability (data not shown). In the present study, FdII has an alternative salt bridge between Arg22 and Glu58 instead of the conservative bond between Arg38 and Glu28. The structure also reveals that in FdII orientation of the {alpha}-helix of Leu23-Glu29 and conformation of the short loop of Lys30-Leu33 connecting this {alpha}-helix with the cluster binding loop are different from those in FdI. Based on this information we produced and characterized FdII mutants with FdI type features: absence of the Arg22 and Glu58 salt bridge (FdII R22T), insertion of a new Tyr residue at position 32 (FdII(Y32)), and insertion of a new Tyr residue combined with construction of a FdI type salt bridge (FdII(Y32) Q38R/S28E). Relative intensities of their A420 due to the presence of the [2Fe-2S] cluster were plotted as a function of time as shown in Fig. 4.



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  		FIG. 4.
Time dependence of the intensities of A420 due to the presence of the [2Fe-2S] cluster for FdII ({circ}), FdII R22T ({triangleup}), FdII(Y32) ({square}), and FdII(Y32)Q38R/S28E ({blacksquare}).

 
The stabilities of the [2Fe-2S] clusters for FdII R22T and FdII(Y32) were significantly decreased, with half-lives of FdII R22T and FdII(Y32) of about 88 and 26 h, respectively. The insertion of Tyr32 perturbed the stability of the [2Fe-2S] cluster more strongly than the cleavage of a salt bridge of Arg22 and Glu58. Stability was restored by introducing a conserved type salt bridge in the FdII(Y32) Q38R/S28E mutant.

Kinetics of Electron Transfer from Fds to FNR—The electron transfer abilities of E. arvense FdI, FdII, and FdII(Y32) Q38R/S28E from Fd to FNR using an NADP+-photoreduction system on chloroplast thylakoid membranes were investigated. In this reaction, NADP+ was reduced via electron transfer from Fd to FNR in the presence of an excess of Fd, reduced by photosystem I. The reduction velocities of NADP+ are plotted as a function of the Fd concentrations at pH 7.5 as shown in Fig. 5.



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  		FIG. 5.
Electron transfer activities of E. arvense FdI ({circ}), FdII ({triangleup}), and FdII(Y32)Q38R/S28E ({square}). Electron transfer activity from Fd to FNR was assayed by measuring the rate of NADP+ photoreduction on chloroplast thylakoid membranes as described under "Experimental Procedures." chl, chlorophyll.

 
Km values of both FdI and FdII were ~0.6 µM, and this similarity is in contrast to our previous measurements of electron transfer in the opposite direction (from FNR to Fd) where the Km value of FdI was around 7 times greater than that of FdII (13). Consistent with our previous measurements, Vmax for electron transfer with FNR was greater with FdI than FdII, although it was 1.3 times greater in electron transfer from Fd to FNR, as compared with 5.5 times greater in the opposite direction (13). The catalytic efficiency, Vmax/Km, of FdI for electron transfer from Fd to FNR is 1.3 times that of FdII, but for the reverse electron transfer from FNR to Fd, catalytic efficiency of FdII is oppositely 1.3 times that of FdI (13). The Km of FdII(Y32) Q38R/S28E was about 1.3 µM and therefore twice those of either FdI or FdII wild types. The Vmax of this FdII mutant was slightly lower (89%) than the FdII wild type. As the Km values of FdI and FdII are almost identical and their Vmax values are also similar, we suspect that this perturbation of the FdII mutants kinetic parameters may be ascribed to secondary effects caused by the introduction of Tyr32 and formation of a salt bridge between Arg38-Glu28 in FdII. Furthermore, we investigated the pH dependence of the FNR reaction with 10 µM (close to Fd saturation at pH 7.5) FdI, FdII, and FdII(Y32) Q38R/S28E (Fig. 6).



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  		FIG. 6.
pH dependence of the electron transfer activity of E. arvense FdI ({circ}), FdII ({triangleup}), and FdII(Y32)Q38R/S28E ({square}). These activities are expressed as the mean ± S.D. of three independent determinations. The solid curves are the theoretical ones (see "Experimental Procedures" for details). chl, chlorophyll.

 
As shown in Fig. 6, all of electron transfer abilities of FdI, FdII, and FdII(Y32) Q38R/S28E show the maximum at pH 7.7. The pH dependences of the reactions with FdI and the FdII mutant between pH 6.4 and 7.7 are well interpreted in terms of the participation of two ionizable groups with pKa 6.0 and 7.4, whereas the pH dependence of the reaction with FdII remains largely unchanged between pH 6.4 and 7.0 and is well interpreted in terms of the participation of single ionizable group with pKa 7.4. All of the abilities decrease rapidly above pH 7.7, and hence the pH dependence above pH 7.7 could not be interpreted with a normal participation of ionizable groups. It should be noted that the number of ionizable group involved in the pH dependence of the reaction with the FdII mutant is the same as that for FdI rather than FdII wild type.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
We have successfully determined the crystal structure of E. arvense FdII at 1.2 Å resolution and compared it to the known crystal structure of E. arvense FdI at 1.8 Å resolution (14). This structure shows that the stabilization mechanism of the [2Fe-2S] cluster differs between FdI and FdII. FdI maintains stability of the [2Fe-2S] cluster through the crucial involvement of a salt bridge between Arg39-Glu28, which is lacking in FdII. The presented data (Figs. 1 and 4) demonstrate that FdII stabilizes the [2Fe-2S] cluster through the deletion of Tyr32 and the formation of a new salt bridge between Arg22-Glu58, compensating for the lack of a salt bridge between Arg39-Glu28. Characterization of FdII mutants confirmed that almost all notable differences between FdI and FdII structures were also related to differences in their stabilization of the [2Fe-2S] cluster.

Our previous x-ray analysis on maize proteins indicated that five intermolecular salt bridges are formed between Fd and leaf FNR (11). E. arvense FdI completely conserves the five residues (Glu28, Arg39, Glu59, Asp64, and Asp65 in E. arvense FdI), which are involved in these intermolecular salt bridges (Fig. 2). However, in E. arvense FdII, the residues corresponding to Glu28 and Arg39 are noncharged (Ser28 and Gln38), and the residue corresponding to Glu59 (Glu58) forms an alternative intramolecular salt bridge with Arg22 indicating Glu58 is not involved in complex formation with FNR. Therefore, we consider the binding mode of FdII with FNR to be considerably different from that of the FdI. We compared kinetic parameters of E. arvense FdI and FdII in electron transfer from Fd to FNR with those for the reverse electron transfer reaction, from FNR to Fd (13) as shown in Table IV.


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  		TABLE IV
Comparison of kinetic parameters of E. arvense FdI and FdII for electron transfer reaction from Fd to FNR with those for reverse electron transfer reaction from FNR to Fd Electron transfer abilities from Fd to FNR were assayed by measuring the rate of NADP+ photoreduction on chloroplast thylakoid membranes, and the reverse electron transfer abilities were done by measuring the rate of cytochrome c reduction in the presence of an excess of NADPH. The values are means ± S.D. of three independent determinations. chl, chlorophyll.

 
It is interesting that the Km and Vmax (or kcat) values of the electron transfer reaction from Fd to FNR vary so little between FdI and FdII, whereas the same kinetic parameters of the reverse reaction are dramatically different. The Km values for electron transfer reactions from Fd to FNR and from FNR to Fd indicate the dissociation constants between reduced Fd and oxidized FNR (or the oxidized FNR·NADP+ complex), and between oxidized Fd and reduced FNR (or the reduced FNR·NADPH complex), respectively. The Km value of FdII for electron transfer from Fd to FNR is somewhat lower than that for the reverse reaction from FNR to Fd. This result may be partly ascribed to the effects of the net charge, because reduced Fd has a further -1 charge, which will favor the binding of reduced Fd to the oxidized FNR·NADP+ complex. On the other hand, The Km value of FdI for electron transfer from Fd to FNR is much lower than that for the reverse reaction from FNR to Fd. This suggests that the interaction between reduced FdI and oxidized FNR induces a more favorable binding mode resulting in an affinity between them that is 18 times higher than that of oxidized FdI with reduced FNR. These findings indicate that the binding mode to FNR apparently differs between FdI and FdII. This could be related to the fact that three of the five Fd residues forming intermolecular salt bridges in the interaction of FdI with FNR have no equivalent in FdII, as described above.

On the basis of a comparison between the crystal structures of oxidized free maize FNR and the oxidized maize FdI·FNR complex (11), we proposed that the interaction of the FdI with FNR induces significant displacement of the loop from Gly82 to Lys91 in FNR, allowing formation of intermolecular salt bridges. These occur between the Lys88 and Lys91 residues of FNR and the Asp66 and Asp65 residues of FdI, respectively. Recently, we have also conducted studies on the dynamic structure of the FdI molecule in solution by NMR, which indicate that the C-terminal region of E. arvense FdI, which is important for binding to Fd-dependent reductases (9, 10), undergoes the conformational change from a flexible extended form to a rigid helical form upon reduction (data not shown). X-ray analysis of the maize FdI·FNR complex had suggested that the Fd C-terminal carboxylate could interact with the side chain of Lys85 of FNR (11), although the Fd C-terminal was highly disordered. These observations provide some insight into the molecular mechanism that results in high affinity between reduced FdI and oxidized FNR as opposed to low affinity between oxidized FdI and reduced FNR.

As shown in Fig. 6, the number of ionizable groups involved in the pH dependence of electron transfer ability for E. arvense FdI is distinct from that of FdII but is replicated with that of FdII(Y32) Q38R/S28E. This finding also indicates that the binding mode of FdI with FNR is different from that of FdII, and that the binding mode of the FdI is partly determined by the Arg39-Glu28 pair. We suspect that the binding modes of further Fd-dependent reductases, such as SiR, NiR, and FTR, are also distinct between E. arvense FdI and FdII. As reported previously, maize FdII lacks a charged residue equivalent to maize FdI Asp65, which is important for the FNR binding. This results in an isoform-specific low rate of NADP+ photoreduction, a reduced donation of electrons to nitrogen assimilation, and a promotion of involvement in cyclic electron flow around photosystem I (8). We therefore consider it likely that E. arvense FdII also varies from E. arvense FdI in its relative distribution of electrons to the numerous Fd-dependent reductases in chloroplasts. It is possible that separate redox networks mediated by FdI and FdII in E. arvense cells are complementary. It is noteworthy that the catalytic efficiency (kcat/Km or Vmax/Km) of FdII in electron transfer toward FNR is slightly lower than that of the FdI but that in contrast the catalytic efficiency of FdII in electron transfer from FNR is slightly higher than that of FdI. Maximum catalytic efficiency in E. arvense chloroplasts would therefore occur if FdII predominantly mediated electron transfer from NADPH-reduced FNR to all other reductases, whereas photoreduction of NADP+, through electron donation from Fd to FNR, is predominantly mediated by FdI. In this way, we hypothesize that FdI and FdII might modulate the distribution of electron flow between different reductive pathways in E. arvense cells. In the future, we plan to investigate the electron transfer abilities of E. arvense FdI and FdII toward Fd-dependent reductases such as FTR, SiR, and NiR and so establish whether the structural variation between FdI and FdII described in this paper results in differential electron donation to different reductive pathways.


   FOOTNOTES
 
* This work was supported in part by Grant-in-aid for Scientific Research 15GS0320 (to T. H.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

§ Present address: Dept. of Life Sciences, Graduate School of Arts and Sciences, the University of Tokyo, 3-8-1 Komaba, Meguro-ku, Tokyo 153-8902, Japan. Back

** To whom correspondence should be addressed: Faculty of Integrated Arts and Sciences, Hiroshima University, 1-7-1 Kagamiyama, Higashi-Hiroshima 739-8521, Japan. Tel.: 81-82-424-6529; Fax: 81-82-424-0757; E-mail: teshi{at}hiroshima-u.ac.jp.

1 The abbreviations used are: Fd, ferredoxin; FNR, ferredoxin-NADP+ oxidoreductase; FTR, ferredoxin-thioredoxin reductase; NiR, nitrite reductase; SiR, sulfite reductase. Back


   ACKNOWLEDGMENTS
 
We are grateful to Professor Kozo Akabori in the Faculty of Integrated Arts and Sciences, Hiroshima University, for encouragement to our research and Professor Atsushi Nakagawa in the Institute for Protein Research, Osaka University for sincere support during x-ray analysis.



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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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