Crystals of Anabaena PCC 7119 ferredoxin-NADP+ reductase

Crystals of ferredoxin-NADP+ reductase (FNR) from the cyanobacterium Anabaena PCC 7119 are grown in the presence of polyethylene glycol 6000 and beta-octyl glucoside. They belong to the hexagonal system. The cell parameters are a = b = 87.8 A, c = 92.7 A, space group P6(1) or P6(5), and a Vm of 3.0 A3/dalton for one molecule of 36,000 daltons per asymmetric unit. These crystals diffract strongly up to 1.9 A and are suitable for X-ray structural studies.     

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

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

Interaction of ferredoxin-NADP+ reductase from Anabaena with its substrates

The interaction of ferredoxin-NADP+ reductase from the cyanobacterium Anabaena variabilis with its substrates, NADP+ and ferredoxin, has been studied by difference absorption spectroscopy. Several structural analogs of NADP+ have been shown to form complexes the stabilities of which are strongly dependent on the ionic strength of the medium. In most cases the binding energy of these complexes and their difference absorption spectra are similar to those reported for the spinach enzyme. However, NADP+ perturbs the absorption spectra of the Anabaena and spinach enzymes in a different way. This difference has been shown to be related to the binding of the nicotinamide ring of NADP+ to the enzymes. These results are interpreted as being due to a different nicotinamide binding site in the two reductases. The enthalpic and entropic components of the Gibbs energy of formation of the NADP+ complex have been estimated. An increase in entropy on NADP+ binding seems to be the main source of stability for the complex. A shift of approximately 40 mV in the redox potential of the couple NADP+/NADPH has been observed to occur upon binding of NADP+ to the oxidized enzyme. This allows us to calculate the binding energy between the reductase and NADPH. The ability of the reductase, ferredoxin, and NADP+ to form a ternary complex indicates that the protein carrier binds to the reductase through a different site than that of the pyridine nucleotide.     

Catalytic mechanism of hydride transfer between NADP+/H and ferredoxin-NADP+ reductase from Anabaena PCC 7119

The mechanism of hydride transfer between Anabaena FNR and NADP+/H was analysed using for the first time stopped-flow photodiode array detection and global analysis deconvolution. The results indicated that the initial spectral changes, occurring within the instrumental dead time upon reaction of FNR with NADP+/H, included not only the initial interaction and complex formation, but also the first subsequent steps of the sequential reactions that involve hydride transfer. Two different charge-transfer complexes formed prior and upon hydride transfer, FNRox-NADPH and FNRrd-NADP+. Detectable amounts of FNRox-NADPH were found at equilibrium, but FNRrd-NADP+ accumulated to a small extent and quickly evolved. The spectral properties of both charge-transfer complexes, for the first time in Anabaena FNR, as well as the corresponding inter-conversion hydride transfer rates were obtained. The need of an adequate initial interaction between NADP+/H and FNR, and subsequent conformational changes, was also established by studying the reactions of two FNR mutants.     

Preparation and crystallization of a cross-linked complex of bovine adrenodoxin and adrenodoxin reductase

Bovine adrenodoxin was cross-linked to adrenodoxin reductase with 1-ethyl-3-(3-dimethyl-aminopropyl) carbodiimide. Mass spectrometry showed the reaction product to be a 1:1 complex of the two proteins with M_r = 64,790 ± 50. The cross-linked complex showed cytochrome c reductase activity and could be crystallized by hanging-drop vapor diffusion. Crystals of the adrenodoxin-adrenodoxin reductase complex are hexagonal, space group P6₁22 or P6₅22, with a = 93.26 Å and c= 612.20 Å and diffract to 2.9 Å resolution at 100 K. Assuming two cross-linked complexes per asymmetric unit yields a reasonable V_M of 2.97 ų/Da. Proteins 28:289–292, 1997. © 1997 Wiley-Liss Inc.     

Probing the role of glutamic acid 139 of Anabaena ferredoxin-NADP+ reductase in the interaction with substrates.

The role of the negative charge of the E139 side-chain of Anabaena Ferredoxin-NADP+ reductase (FNR) in steering appropriate docking with its substrates ferredoxin, flavodoxin and NADP+/H, that leads to efficient electron transfer (ET) is analysed by characterization of several E139 FNR mutants. Replacement of E139 affects the interaction with the different FNR substrates in very different ways. Thus, while E139 does not appear to be involved in the processes of binding and ET between FNR and NADP+/H, the nature and the conformation of the residue at position 139 of Anabaena FNR modulates the precise enzyme interaction with the protein carriers ferredoxin (Fd) and flavodoxin (Fld). Introduction of the shorter aspartic acid side-chain at position 139 produces an enzyme that interacts more weakly with both ET proteins. Moreover, the removal of the charge, as in the E139Q mutant, or the charge-reversal mutation, as in E139K FNR, apparently enhances additional interaction modes of the enzyme with Fd, and reduces the possible orientations with Fld to more productive and stronger ones. Hence, removal of the negative charge at position 139 of Anabaena FNR produces a deleterious effect in its ET reactions with Fd whereas it appears to enhance the ET processes with Fld. Significantly, a large structural variation is observed for the E139 side-chain conformer in different FNR structures, including the E139K mutant. In this case, a positive potential region replaces a negative one in the wild-type enzyme. Our observations further confirm the contribution of both attractive and repulsive interactions in achieving the optimal orientation for efficient ET between FNR and its protein carriers.     

Purification of ferredoxin-NADP+ reductase, flavodoxin and ferredoxin from a single batch of the cyanobacterium Anabaena PCC 7119.

Methods are described for the simultaneous isolation of ferredoxin-NADP+ reductase, ferredoxin and flavodoxin from large quantities of the cyanobacterium Anabaena PCC 7119 allowing the use of a single batch of cells. The ultraviolet-visible spectra and the extinction coefficients of ferredoxin-NADP+ reductase and ferredoxin were determined. The purification procedure also yields enriched fractions of phycobiliproteins and cytochrome c553.     

Identification of a cross-linked peptide of a covalent complex between adrenodoxin reductase and adrenodoxin.

A cross-linked complex between bovine NADPH-adrenodoxin reductase (AR) and adrenodoxin (AD) was prepared with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and purified, as described previously [Hara, T. & Kimura, T. (1989) J. Biochem. 105, 594-600]. The covalent complex was S-pyridylethylated and digested with lysylendopeptidase, and the resulting peptides were separated by reversed-phase HPLC to identify the cross-linked peptide. Comparison of the HPLC chromatograms of the peptides showed that (i) two tandem peptides (K-4 and K-5) from AD and a peptide (K-1) from AR were missing in the chromatogram of the peptides of the covalent complex and (ii) a single new peak was observed in the chromatogram of the peptides from the covalent complex. Amino acid composition and sequence analyses showed that the newly observed peptide was a covalently cross-linked peptide formed between a peptide K-4-K-5 (Ile-25-Lys-98) derived from AD and a peptide K-1 (Ser-1-Lys-27) derived from AR, in which an amide bond had been formed between the epsilon-amino group of Lys-66 in AD and the gamma-carboxyl group of Glu-4 in AR. These results indicate that the binding site of AR with AD is localized in the amino-terminal part of AR and that of AD with AR is localized around Lys-66 of AD. The six clustered basic amino acid residues (His-24, Lys-27, His-28, His-29, Arg-31, and His-33) present in the amino-terminal portion of AR and the eight clustered acidic amino acid residues (Glu-65, Glu-68, Asp-72, Glu-73, Glu-74, Asp-76, Asp-79, and Asp-86) present in the middle part of AD may play an important role in the complex formation.     

Effects of chemical modification of Anabaena flavodoxin and ferredoxin-NADP+ reductase on the kinetics of interprotein electron transfer reactions.

The influence of chemical modification of arginine residues (using phenylglyoxal) in ferredoxin-NADP+ reductase (FNR), and of carboxyl groups (using glycine ethyl ester) in flavodoxin (Fld), on the kinetics of electron transfer between FNR and Fld, and between ferredoxin (Fd) and FNR, was examined using laser flash photolysis methods. All proteins were obtained from the cyanobacterium Anabaena PCC7119. Reduction by laser-generated 5-deazariboflavin semiquinone of the FAD moiety of phenylglyoxal-modified FNR occurred with a second-order rate constant 2.5-fold smaller than that obtained for reduction of native FNR, indicating either a small degree of steric hindrance of the cofactor, or a decrease in its redox potential, upon chemical modification. In contrast, no changes were found in the kinetics of reduction of the FMN cofactor of Fld modified by glycine ethyl ester as compared with the native protein. The observed rate constants for reoxidation of Fdred (reduced Fd) by FNRox (oxidized FNR) were dramatically decreased (approximately 100-fold) when phenylglyoxal-modified FNR was used. In contrast to the reaction involving the native proteins, no ionic strength effects on kobs values were found. These results, and those obtained upon varying the protein concentration, indicate that the rate constant for complex formation and the attractive electrostatic interaction between the two proteins were greatly diminished by chemical modification of arginine residues of FNR. When phenylglyoxal-modified FNRsq (FNR semiquinone) was used to reduce Fldox (oxidized Fld), similar inhibitory effects were observed. In this case, the limiting first-order rate constant for Fldsq (Fld semiquinone) formation via intracomplex electron transfer from FNRsq was approximately 12-fold smaller than that obtained for the native FNR (600 s-1 vs 7000 s-1). Again, ionic strength effects were diminished. The glycine-ethyl-ester-modified Fld yielded a limiting first-order rate constant for intracomplex electron transfer from FNRsq to Fldox which was approximately 7-fold smaller (1000 s-1) than that obtained with native Fld, and ionic strength effects were again diminished. These results indicate that complex formation can still occur between modified FNR and native Fld, and between native FNR and modified Fld, but that the geometry of these complexes is altered so as to decrease the effectiveness of interprotein electron transfer. The results are discussed in terms of the specific structural features of the proteins involved.     

Oxidation-reduction potentials of ferredoxin-NADP+ reductase and flavodoxin from Anabaena PCC 7119 and their electrostatic and covalent complexes.

The oxidation-reduction potentials of ferredoxin-NADP+ reductase and flavodoxin from the cyanobacterium Anabaena PCC 7119 were determined by potentiometry. The potentials at pH 7 for the oxidized flavodoxin/flavodoxin semiquinone couple (E2) and the flavodoxin semiquinone/hydroquinone couple (E1) were -212 mV and -436 mV, respectively. E1 was independent of pH above about pH 7, but changed by approximately -60 mV/pH below about pH 6, suggesting that the fully reduced protein has a redox-linked pKa at about 6.1, similar to those of certain other flavodoxins. E2 varied by -50 mV/pH in the range pH 5-8. The redox potential for the two-electron reduction of ferredoxin-NADP+ reductase was -344 mV at pH 7 (delta Em = -30 mV/pH). In the 1:1 electrostatic complex of the two proteins titrated at pH 7, E2 was shifted by +8 mV and E1 was shifted by -25 mV; the shift in potential for the reductase was +4 mV. The potentials again shifted following treatment of the electrostatic complex with a carbodiimide, to covalently link the two proteins. By comparison with the separate proteins at pH 7, E2 for flavodoxin shifted by -21 mV and E1 shifted by +20 mV; the reductase potential shifted by +2 mV. The potentials of the proteins in the electrostatic and covalent complexes showed similar pH dependencies to those of the individual proteins. Qualitatively similar changes occurred when ferredoxin-NADP+ reductase from Anabaena variabilis was complexed with flavodoxin from Azotobacter vinelandii. The shifts in redox potential for the complexes were used with previously determined values for the dissociation constant (Kd) of the electrostatic complex of the two oxidised proteins, in order to estimate Kd values for the interaction of the different redox forms of the proteins. The calculations showed that the electrostatic complexes, formed when the proteins differ in their redox states, are stronger than those formed when both proteins are fully oxidized or fully reduced.     

Purification and catalytic properties of a cross-linked complex between adrenodoxin reductase and adrenodoxin

A stable covalent complex was prepared by cross-linking adrenodoxin reductase with adrenodoxin using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide. The covalent complex was purified extensively until free components were removed completely. The major component of the complex had a molecular weight of 63 kDa, which corresponds to a 1:1 stoichiometric complex between adrenodoxin reductase and adrenodoxin. NADPH-cytochrome c reduction activity of the covalent complex was comparable to that of an equimolar mixture of adrenodoxin reductase and adrenodoxin (native complex), and the NADPH-ferricyanide reduction activity of the complex was equal to that of the native one. In contrast to the native complex, the covalent complex produced much less superoxide upon NADPH-oxidation, and the covalent complex was found to be more stable than the native complex, suggesting that the complex state is more favorable for catalysis. From these results, we conclude that the adrenodoxin molecule does not need to dissociate from the complex during electron transfer from NADPH to cytochrome c.     

Domain exchange between isoforms of ferredoxin-NADP+ reductase produces a functional enzyme

Two isoforms of ferredoxin-NADP(+) reductase (FNR) exist in higher plants, the leaf (or photosynthetic) and the root (or non-photosynthetic) isoform, which have 48% amino acid sequence identity and display specific structural and functional features. With the aim to gain further insight into the structure-function relationship of this enzyme, we designed two novel chimeric flavoenzymes by swapping the structural domains between the leaf and the root isoforms. Characterization of the chimeras would allow dissection of the contribution of the individual domains to catalysis. The chimera obtained by grafting together the FAD-binding domain of the root-isoform and the NADP-binding domain of the leaf-isoform was inactive when expressed in Escherichia coli. On the other hand, the chimera assembled in the opposite way (leaf FAD-binding domain and root NADP-binding domain) was functional and was produced in the bacterial host to a level threefold higher than that of the parent enzymes. The protein was purified and found to be as stable as the natural isoforms. Limited proteolysis excluded the presence in the chimera of misfolded regions. The affinity of the chimera for ferredoxin I (Fd I) was similar to that of the leaf isoform, although interprotein electron-transfer was partially impaired. As occurs with the root isoform, the chimera bound NADP(+) with high affinity, while spectroscopic evidence suggested that the conformation adopted by the nicotinamide moiety bound to the chimera was similar to that observed in the leaf enzyme. Interestingly, the chimera, by combining favorable features from both parent isoforms, acquired a catalytic efficiency (k(cat)/K(m)), as an NADPH-dependent diaphorase, higher than those of both the root ( approximately 2-fold) and the leaf enzyme ( approximately 5-fold). Thus, molecular breeding between isozymes has improved the catalytic properties of FNR.     

Enzymatic oxidation of NADP+ to its 4-oxo derivative is a side-reaction displayed only by the adrenodoxin reductase type of ferredoxin-NADP+ reductases

We have previously shown that Mycobacterium tuberculosis FprA, an NADPH-ferredoxin reductase homologous to mammalian adrenodoxin reductase, promotes the oxidation of NADP(+) to its 4-oxo derivative 3-carboxamide-4-pyridone adenine dinucleotide phosphate [Bossi RT, Aliverti A, Raimondi D, Fischer F, Zanetti G, Ferrari D, Tahallah N, Maier CS, Heck AJ, Rizzi M et al. (2002) Biochemistry41, 8807-8818]. Here, we provide a detailed study of this unusual enzyme reaction, showing that it occurs at a very slow rate (0.14 h(-1)), requires the participation of the enzyme-bound FAD, and is regiospecific in affecting only the C4 of the NADP nicotinamide ring. By protein engineering, we excluded the involvement in catalysis of residues Glu214 and His57, previously suggested to be implicated on the basis of their localization in the three-dimensional structure of the enzyme. Our results substantiate a catalytic mechanism for 3-carboxamide-4-pyridone adenine dinucleotide phosphate formation in which the initial and rate-determining step is the nucleophilic attack of the nicotinamide moiety by an active site water molecule. Whereas plant-type ferredoxin reductases were unable to oxidize NADP(+), the mammalian adrenodoxin reductase also catalyzed this unusual reaction. Thus, the 3-carboxamide-4-pyridone adenine dinucleotide phosphate formation reaction seems to be a peculiar feature of the mitochondrial type of ferredoxin reductases, possibly reflecting conserved properties of their active sites. Furthermore, we showed that 3-carboxamide-4-pyridone adenine dinucleotide phosphate is good ligand and a competitive inhibitor of various dehydrogenases, making this nucleotide analog a useful tool for the characterization of the cosubstrate-binding site of NADPH-dependent enzymes.     

Complex-forming properties of butanedione-modified ferredoxin-NADP+ reductase with NADP+ and ferredoxin.

The plastidic ferredoxin-NADP⁺ reductase from the xanthophycean alga Bumilleriopsis forms a stoichiometric 1:1 complex with ferredoxin and NADP⁺ which is demonstrated by difference spectra of both complexes. Butanedione modification of the flavoprotein results in loss of its enzymatic activities (transhydrogenase and diaphorase) concurrently with its capability to form a complex with NADP⁺, whereas the ferredoxin-binding site is practically not influenced by the modifying reagent and complex formation is still possible. It is assumed, therefore, that butanedione specifically reacts with the arginine residue of the protein involved in binding of pyridine nucleotides at the active site. Further, the data presented strongly support the previous proposal of different binding sites for ferredoxin and pyridine nucleotides at the reductase.     

Identification of specific carboxyl groups on Anabaena PCC 7119 flavodoxin which are involved in the interaction with ferredoxin-NADP+ reductase.

Flavodoxin from the nitrogen-fixing cyanobacteria Anabaena PCC 7119 forms an electron-transfer complex with ferredoxin--NADP+ reductase (FNR) from the same organism. The complex is mainly governed by electrostatic interactions between side-chain amino groups of the reductase and carboxyl residues of flavodoxin. In order to localize the binding site on flavodoxin, chemical modification of its carboxyl groups has been carried out. Treatment of flavodoxin with a water-soluble carbodiimide, N-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), in the presence of a nucleophile, glycine ethyl ester, caused a time-dependent modification of the protein that is responsible for the loss of its ability to participate as electron carrier in the photoreduction of NADP+ by chloroplast membranes, and also in NADPH--cytochrome-c reductase activity, by about 85%. Nevertheless, the ability of flavodoxin to receive electrons from the reducing side of photosystem I was much less affected. The inhibition was enhanced at low pH, suggesting that carboxylic acid groups were the target of chemical modification. Treated flavodoxin failed to form covalent complexes with FNR and the dissociation constant for the non-covalent complex with FNR was fourfold higher. After tryptic digestion of a sample of flavodoxin modified by EDC in the presence of [1-14C]glycine ethyl ester, two major radioactive peptides were isolated. The first protein fragment contained three carboxylic residues (Asp123, Asp126 and Asp129), corresponding to the region where long-chain flavodoxins show an insert compared to short-chain flavodoxins. The second peptide corresponded to a similar region, either in the amino acid sequence or in the three-dimensional structure of the protein and also containing three carboxyl groups (Asp144, Glu145 and Asp146). Four of these carboxyl groups (Asp123, Asp126, Asp144 and Asp146) are highly conserved in all long-chain flavodoxins, suggesting that they could play an essential role in substrate recognition.     

Inhibition of adrenodoxin reductase by NADP+ and NADPH

Adrenodoxin reductase (EC 1.18.1.2) catalyzes the oxidation of NADPH by 1.4-benzoquinone. The catalytic constant of this reaction at pH 7.0 is equal to 25-28 s-1. NADP+ acts as the mixed-type nonlinear inhibitor of enzyme increasing Km of NADPH and decreasing catalytic constant. NADP+ and NADPH act as mutually exclusive inhibitors relative to reduced adrenodoxin reductase. The patterns of 2',5'-ADP inhibition are analogous to that of NADP+. These data support the conclusion about the existence of second nicotinamide coenzyme binding centre in adrenodoxin reductase.     

Purification and properties of ferredoxin-NADP+ oxidoreductase from the nitrogen-fixing cyanobacteria Anabaena variabilis

The isolation and characterization of ferredoxin-NADP+ -oxidoreductase from Anabaena variabilis, a nitrogen-fixing, filamentous cyanobacterium, is described. Purified enzyme was obtained in four steps with a 55% yield and 300-fold purification utilizing chromatographic separations on DEAE-cellulose and Cibacron Blue-Sepharose columns. The enzyme is quite similar but not identical to the spinach enzyme as judged by isoelectric focusing, molecular weight determination, and amino acid composition. N-terminal sequence analysis allowed identification of 28 of the first 33 residues. Alignment with the corresponding sequences from spinach and Spirulina FNR preparations was possible. A higher degree of homology was found with the Spirulina enzyme than with the spinach enzyme. Small differences with the spinach enzyme were also shown by absorption and circular dichroism spectral measurements. Oxidation-reduction potential measurements of the bound FAD coenzyme show an Em = -320 mV at pH 7 for the two-electron process. Complex formation between the reductase and ferredoxin from the same organism was observed by difference absorption spectroscopy with a Kd = 4 microM. Similar Kd and difference absorption properties were observed on complex formation with spinach ferredoxin.