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PubMed1999

Complex formation between Azotobacter vinelandii ferredoxin I and its physiological electron donor NADPH-ferredoxin reductase.

In Azotobacter vinelandii, deletion of the fdxA gene, which encodes ferredoxin I (FdI), leads to activation of the expression of the fpr gene, which encodes NADPH-ferredoxin reductase (FPR). In order to investigate the relationship of these two proteins further, the interactions of the two purified proteins have been examined. AvFdI forms a specific 1:1 cross-linked complex with AvFPR through ionic interactions formed between the Lys residues of FPR and Asp/Glu residues of FdI. The Lys in FPR has been identified as Lys258, a residue that forms a salt bridge with one of the phosphate oxygens of FAD in the absence of FdI. UV-Vis and circular dichroism data show that on binding FdI, the spectrum of the FPR flavin is hyperchromatic and red-shifted, confirming the interaction region close to the FAD. Cytochrome c reductase assays and electron paramagnetic resonance data show that electron transfer between the two proteins is pH-dependent and that the [3Fe-4S]+ cluster of FdI is specifically reduced by NADPH via FPR, suggesting that the [3Fe-4S] cluster is near FAD in the complex. To further investigate the FPR:FdI interaction, the electrostatic potentials for each protein were calculated. Strongly negative regions around the [3Fe-4S] cluster of FdI are electrostatically complementary with a strongly positive region overlaying the FAD of FPR, centered on Lys258. These proposed interactions of FdI with FPR are consistent with cross-linking, peptide mapping, spectroscopic, and electron transfer data and strongly support the suggestion that the two proteins are physiological redox partners.

Amino Acid Sequence↗
PubMed2004

A structural basis of Equisetum arvense ferredoxin isoform II producing an alternative electron transfer with ferredoxin-NADP+ reductase.

We have determined the crystal structure, at 1.2-A resolution, of Equisetum arvense ferredoxin isoform II (FdII), which lacks residues equivalent to Arg(39) and Glu(28) 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 Arg(22) and Glu(58). This results in a different relative orientation of the alpha-helix formed by Leu(23)-Glu(29) 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 K(m) and V(max) 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.

Amino Acid Sequence↗
PubMed1999

Electrostatic forces involved in orienting Anabaena ferredoxin during binding to Anabaena ferredoxin:NADP+ reductase: site-specific mutagenesis, transient kinetic measurements, and electrostatic surface potentials.

Transient absorbance measurements following laser flash photolysis have been used to measure the rate constants for electron transfer (et) from reduced Anabaena ferredoxin (Fd) to wild-type and seven site-specific charge-reversal mutants of Anabaena ferredoxin:NADP+ reductase (FNR). These mutations have been designed to probe the importance of specific positively charged amino acid residues on the surface of the FNR molecule near the exposed edge of the FAD cofactor in the protein-protein interaction during et with Fd. The mutant proteins fall into two groups: overall, the K75E, R16E, and K72E mutants are most severely impaired in et, and the K138E, R264E, K290E, and K294E mutants are impaired to a lesser extent, although the degree of impairment varies with ionic strength. Binding constants for complex formation between the oxidized proteins and for the transient et complexes show that the severity of the alterations in et kinetics for the mutants correlate with decreased stabilities of the protein-protein complexes. Those mutated residues, which show the largest effects, are located in a region of the protein in which positive charge predominates, and charge reversals have large effects on the calculated local surface electrostatic potential. In contrast, K138, R264, K290, and K294 are located within or close to regions of intense negative potential, and therefore the introduction of additional negative charges have considerably smaller effects on the calculated surface potential. We attribute the relative changes in et kinetics and complex binding constants for these mutants to these characteristics of the surface charge distribution in FNR and conclude that the positively charged region of the FNR surface located in the vicinity of K75, R16, and K72 is especially important in the binding and orientation of Fd during electron transfer.

Anabaena↗
PubMed2005

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

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

Catalysis↗
PubMed1999

Ferredoxin-NADP(+) reductase uses the same site for the interaction with ferredoxin and flavodoxin.

The enzyme ferredoxin-NADP(+) reductase (FNR) forms a 1 : 1 complex with ferredoxin (Fd) or flavodoxin (Fld) that is stabilised by both electrostatic and hydrophobic interactions. The electrostatic interactions occur between acidic residues of the electron transfer (ET) protein and basic residues on the FNR surface. In the present study, several charge-reversal mutants of FNR have been prepared at the proposed site of interaction of the ET protein: R16E, K72E, K75E, K138E, R264E, K290E and K294E. All of these mutants have been assayed for reactivity with Fd and Fld using steady-state and stopped-flow kinetics. Their abilities for complex formation with the ET proteins have also been tested. The data presented here indicate that the mutated residues situated within the FNR FAD-binding domain are more important for achieving maximal ET rates, either with Fd or Fld, than those situated within the NADP(+)-binding domain, and that both ET proteins occupy the same region for the interaction with the reductase. In addition, each individual residue does not appear to participate to the same extent in the different processes with Fd and Fld.

Anabaena↗
PubMed1999

The solution structure of a [3Fe-4S] ferredoxin: oxidised ferredoxin II from Desulfovibrio gigas.

The use of standard 2D NMR experiments in combination with 1D NOE experiments allowed the assignment of 51 of the 58 spin systems of oxidised [3Fe4S] ferredoxin isolated from Desulfovibrio gigas. The NMR solution structure was determined using data from 1D NOE and 2D NOESY spectra, as distance constraints, and information from the X-ray structure for the spin systems not detected by NMR in torsion angle dynamics calculations to produce a family of 15 low target function structures. The quality of the NMR family, as judged by the backbone r.m.s.d. values, was good (0.80 A), with the majority of phi/psi angles falling within the allowed region of the Ramachandran plot. A comparison with the X-ray structure indicated that the overall global fold is very similar in solution and in the solid state. The determination of the solution structure of ferredoxin II (FdII) in the oxidised state (FdIIox) opens the way for the determination of the solution structure of the redox intermediate state of FdII (FdII(int)), for which no X-ray structure is available.

Amino Acid Sequence↗
PubMed1999

Engineering and biochemical characterization of the rat microsomal cytochrome P4501A1 fused to ferredoxin and ferredoxin-NADP(+) reductase from plant chloroplasts.

Fusion proteins of rat cytochrome P4501A1 with maize ferredoxin I (Fd) and pea ferredoxin NADP(+) reductase (FNR), the last electron transfer proteins of the photosynthetic channel in plant chloroplasts, were obtained by gene fusion in the yeast expression vector pAAH5N. The encoded fusion proteins P4501A1-Fd, P4501A1-FNR, P4501A1-Fd-FNR and P4501A1-FNR-Fd were produced in microsomes of the yeast Saccharomyces cerevisiae AH22. Enzymatic assays were carried out in vitro with the isolated microsomes. P4501A1-Fd-FNR and P4501A1-FNR-Fd were found to catalyze P450-monooxygenase activities towards 7-ethoxycoumarin and the herbicide chlortoluron. P4501A1-Fd-FNR was the most efficient enzyme as measured in vitro in ferricyanide and cytochrome c reductions, as well as P450-monooxygenase assays. Apparent K(m) and k(cat) of P4501A1-Fd-FNR were 70 microM and 7800 min(-1) for NADPH, 13.2 microM and 51.1 min(-1) for 7-ethoxycoumarin, and 21.3 microM and 23. 8 min(-1) for the herbicide chlortoluron, respectively. Fd in P4501A1-Fd-FNR fusion enzyme was found to be a limiting factor compared to P4501A1 fused to the yeast NADPH-cytochrome P450 reductase, an artificial enzyme described previously. The efficiency of electron transfer in the P4501A1 fusion proteins and a possible in vivo molecular coupling of Fd and FNR with microsomal cytochrome P4501A1 produced in plant chloroplasts are discussed.

7-Alkoxycoumarin O-Dealkylase↗
PubMed1994

Calculation of the redox potentials of iron-sulfur proteins: the 2-/3-couple of [Fe4S*4Cys4] clusters in Peptococcus aerogenes ferredoxin, Azotobacter vinelandii ferredoxin I, and Chromatium vinosum high-potential iron protein.

Calculations of the redox potentials of the 2-/3-couples of [Fe4S*4Cys4] clusters in the iron-sulfur proteins Peptococcus aerogenes ferredoxin (PaFd), Azotobacter vinelandii ferredoxin I (AvFdI) and Chromatium vinosum high potential iron protein (CvHiPIP) based on the Protein Dipoles Langevin Dipoles (PDLD) method are reported. The structures of these proteins have been determined by X-ray crystallography; in the case of PaFd the structure has recently been revised due to a change in the sequence close to Cluster II. The large differences between the potentials of the [Fe4S*4Cys4] clusters of PaFd and AvFdI and the potential of the [Fe4S*4Cys4] cluster of CvHiPIP are successfully modeled and originate principally in differences in the configuration of main-chain amide groups near the clusters. The small difference between the potentials of PaFd and AvFdI is also satisfactorily modeled in the case of Cluster I of PaFd. Solvent dipoles close to the cluster in PaFd are an important contributor to its higher potential. The two X-ray structures of PaFd yield similar results for Cluster I of PaFd. In contrast, the results for Cluster II differ substantially; for reasons not yet clear, the recently revised structure leads to results in worse agreement with experiment.

Amino Acid Sequence↗
PubMed2004

Multiple orientations in a physiological complex: the pyruvate-ferredoxin oxidoreductase-ferredoxin system.

Ferredoxin I from Desulfovibrio africanus (Da FdI) is a small acidic [4Fe-4S] cluster protein that exchanges electrons with pyruvate-ferredoxin oxidoreductase (PFOR), a key enzyme in the energy metabolism of anaerobes. The thermodynamic properties and the electron transfer between PFOR and either native or mutated FdI have been investigated by microcalorimetry and steady-state kinetics, respectively. The association constant of the PFOR-FdI complex is 3.85 x 10(5) M(-1), and the binding affinity has been found to be highly sensitive to ionic strength, suggesting the involvement of electrostatic forces in formation of the complex. Surprisingly, the punctual or combined neutralizations of carboxylate residues surrounding the [4Fe-4S] cluster slightly affect the PFOR-FdI interaction. Furthermore, hydrophobic residues around the cluster do not seem to be crucial for the PFOR-FdI system activity; however, some of them play an important role in the stability of the FeS cluster. NMR restrained docking associated with site-directed mutagenesis studies suggested the presence of various interacting sites on Da FdI. The modification of additional acidic residues at the interacting interface, generating a FdI pentamutant, evidenced at least two distinct FdI binding sites facing the distal [4Fe-4S] cluster of the PFOR. We also used a set of various small acidic partners to investigate the specificity of PFOR toward redox partners. The remarkable flexibility of the PFOR-FdI system supports the idea that the specificity of the physiological complex has probably been "sacrificed" to improve the turnover rate and thus the efficiency of bacterial electron transfer.

Amino Acid Sequence↗
PubMed1997

Iron-sulfur cluster cysteine-to-serine mutants of Anabaena -2Fe-2S- ferredoxin exhibit unexpected redox properties and are competent in electron transfer to ferredoxin:NADP+ reductase.

The reduction potentials and the rate constants for electron transfer (et) to ferredoxin:NADP+ reductase (FNR) are reported for site-directed mutants of the [2Fe-2S] vegetative cell ferredoxin (Fd) from Anabaena PCC 7120, each of which has a cluster ligating cysteine residue mutated to serine (C41S, C46S, and C49S). The X-ray crystal structure of the C49S mutant has also been determined. The UV-visible optical and CD spectra of the mutants differ from each other and from wild-type (wt) Fd. This is a consequence of oxygen replacing one of the ligating cysteine sulfur atoms, thus altering the ligand --> Fe charge transfer transition energies and the chiro-optical properties of the chromophore. Each mutant is able to rapidly accept an electron from deazariboflavin semiquinone (dRfH.) and to transfer an electron from its reduced form to oxidized FNR although all are somewhat less reactive (30-50%) toward FNR and are appreciably less stable in solution than is wt Fd. Whereas the reduction potential of C46S (-381 mV) is not significantly altered from that of wt Fd (-384 mV), the potential of the C49S mutant (-329 mV) is shifted positively by 55 mV, demonstrating that the cluster potential is sensitive to mutations made at the ferric iron in reduced [2Fe-2S] Fds with localized valences. Despite the decrease in thermodynamic driving force for et from C49S to FNR, the et rate constant is similar to that measured for C46S. Thus, the et reactivity of the mutants does not correlate with altered reduction potentials. The et rate constants of the mutants also do not correlate with the apparent binding constants of the intermediate (Fdred:FNRox) complexes or with the ability of the prosthetic group to be reduced by dRfH.. Furthermore, the X-ray crystal structure of the C49S mutant is virtually identical to that of wt Fd. We conclude from these data that cysteine sulfur d-orbitals are not essential for et into or out of the iron atoms of the cluster and that the decreased et reactivity of these Fd mutants toward FNR may be due to small changes in the mutual orientation of the proteins within the intermediate complex and/or alterations in the electronic structure of the [2Fe-2S] cluster.

Anabaena↗
PubMed2002

CYP119 plus a Sulfolobus tokodaii strain 7 ferredoxin and 2-oxoacid:ferredoxin oxidoreductase constitute a high-temperature cytochrome P450 catalytic system.

The cytochrome P450 superfamily of enzymes catalyzes a broad range of oxidative processes involved in the metabolism of fatty acids, biosynthesis of sterols, and elimination of drugs and xenobiotics. Application of the unique properties of P450 enzymes as fine biocatalysts in biotechnology is limited due to their thermal instability and the requirement for auxiliary electron-donor proteins and cofactors. CYP119, a thermophilic P450 enzyme from Sulfolobus solfataricus, was characterized some time ago, but no high-temperature redox partners have been available for it. Here we report reconstitution of CYP119 with a novel high-temperature electron-donor system consisting of a ferredoxin and 2-oxoacid:ferredoxin oxidoreductase from Sulfolobus tokodaii strain 7 that, unlike all other known P450 electron-donor partners, utilizes coenzyme-A and pyruvic acid rather than NADH or NADPH as the source of electrons. The oxidation of lauric acid by the reconstituted system increased 16-fold as the temperature increased from 25 to 70 degrees C and was functional for more than 30 min at the higher temperature. This first in vitro high-temperature P450 catalytic system is a key step in the development of practical high-temperature monooxygenase systems.

Archaeal Proteins↗
PubMed1983

Reduction of methemoglobins M Hyde Park, M Saskatoon, and M Milwaukee by ferredoxin and ferredoxin-nicotinamide adenine dinucleotide phosphate reductase system.

The reduction of hemoglobins (Hb) M such as Hb M Iwate, Hb M Boston, Hb M Hyde Park, Hb M Saskatoon, and Hb M Milwaukee by the ferredoxin and ferredoxin-NADP reductase system was studied systematically under anaerobic conditions. The enzyme system could not reduce the abnormal chains in methemoglobin M with an alpha chain anomaly but effectively converted the methemoglobin M with a beta chain anomaly to the fully reduced form. During the reduction of the methemoglobin M with a beta chain anomaly, the spectra showed a shift of the initial isosbestic points, indicating the possible formation of intermediate hemoglobins in the partially reduced state. On the reduction mode of the methemoglobin M, however, it was classified into three types. 1) Only normal chains were reduced (Hb M Iwate and Hb M Boston). 2) Sequential reduction from normal to abnormal chains occurred (Hb M Milwaukee and Hb M Hyde Park). 3) Normal chains were preferentially reduced, but the reduction of abnormal chains also started at the same rate when the reduction of normal ones had proceeded halfway (Hb M Saskatoon). These differences are discussed in relation to the redox potential of each abnormal chain in methemoglobin M.

Ferredoxins↗
PubMed1998

The delta-subunit of pyruvate ferredoxin oxidoreductase from Pyrococcus furiosus is a redox-active, iron-sulfur protein: evidence for an ancestral relationship with 8Fe-type ferredoxins.

Pyruvate ferredoxin oxidoreductase (POR) from the hyperthermophilic archaeon Pyrococcus furiosus (Pf) catalyzes the final oxidative step in carbohydrate fermentation in which pyruvate is oxidized to acetyl-CoA and CO2, coupled to the reduction of ferredoxin (Fd). POR is composed of two 'catalytic units' of molecular mass approximately 120 kDa. Each unit consists of four subunits, alpha beta gamma delta, with masses of approximately 44, 36, 20, and 12 kDa, respectively, and contains at least two [4Fe-4S] clusters. The precise mechanism of catalysis and the role of the individual subunits are not known. The gene encoding the delta-subunit of Pf POR has been expressed in E. coli, and the protein was purified after reconstitution with iron and sulfide. The reconstituted delta-subunit (recPOR-delta) is monomeric with a mass of 11 879 +/- 1.2 Da as determined by mass spectrometry, in agreement with that predicted from the gene sequence. Purified recPOR-delta contains 8 Fe mol/mol and remained intact when incubated at 85 degreesC for 2 h, as judged by its visible absorption properties. The reduced form of the protein exhibited an EPR spectrum characteristic of two, spin-spin interacting [4Fe-4S]1+ clusters. When compared with the EPR properties of the reduced holoenzyme, the latter was shown to contain a third [4Fe-4S]1+ cluster in addition to the two within the delta-subunit. The reduction potential of the two 4Fe clusters in isolated recPOR-delta (-403 +/- 8 mV at pH 8.0 and 24 degreesC) decreased linearly with temperature (-1.55 mV/ degreesC) up to 82 degreesC. RecPOR-delta replaced Pf Fd as an in vitro electron carrier for two oxidoreductases from Pf, POR and Fd:NADP oxidoreductase, and the POR holoenzyme displayed a higher apparent affinity for its own subunit (apparent Km = 1.0 microM at 80 degreesC) than for Fd (apparent Km = 4.4 microM). The molecular and spectroscopic properties and amino acid sequence of the isolated delta-subunit suggest that it evolved from an 8Fe-type Fd by the addition of approximately 40 residues at the N-terminus, and that this extension enabled it to interact with additional subunits within POR.

Amino Acid Sequence↗
PubMed1985

Effect of Ferredoxin on the Diaphorase Activity of Cyanobacterial Ferredoxin-NADP Reductase.

The interaction of ferredoxin-NADP reductase (FNR) and ferredoxin (Fd) results in an enhanced rate of reaction and a shift of the pH optimum for the FNR-mediated diaphorase reaction. Low concentrations of NaCl (<100 millimolar), favorable for formation of the FNR:Fd complex, further magnify the alteration of the diaphorase reaction; the activity is enhanced 3-fold and pH optimum is shifted from 9.5 to 7.8. The Fd-stimulated diaphorase activity of FNR may result either from a conformational change of the enzyme and/or from a transition from a two electron to a one electron reaction.

Journal Article↗
PubMed1995

Molecular characterization of the genes encoding the tungsten-containing aldehyde ferredoxin oxidoreductase from Pyrococcus furiosus and formaldehyde ferredoxin oxidoreductase from Thermococcus litoralis.

The hyperthermophilic archaea Pyrococcus furiosus and Thermococcus litoralis contain the tungstoenzymes aldehyde ferredoxin oxidoreductase, a homodimer, and formaldehyde ferredoxin oxidoreductase, a homotetramer. herein we report the cloning and sequencing of the P. furiosus gene aor (605 residues; M(r), 66,630) and the T. litoralis gene for (621 residues; M(r), 68,941).

Aldehyde Oxidoreductases↗
PubMed1982

Spinach siroheme enzymes: Isolation and characterization of ferredoxin-sulfite reductase and comparison of properties with ferredoxin-nitrite reductase.

Sulfite reductase (SiR) has been purified to homogeneity from spinach leaves. Two forms of the enzyme were separated by hydroxylapatite chromatography. One, with subunit Mr 69 000 appears to be proteolytically cleaved to give rise to the other, with subunit Mr 63 000, during the purification procedure. The two species have identical catalytic activities (on a per heme basis) when reduced methylviologen (MV+) or ferredoxin (Fdr) is used as electron donor for sulfite reduction, and they exhibit nearly identical optical and EPR spectra. Both enzyme forms exist in 50 mM phosphate buffer (pH 7.7) primarily as dimers at 20 degrees C. Spinach SiR contains 1 mol of siroheme and one Fe4S4 center per subunit. The heme iron is the high spin Fe3+ state in the enzyme as isolated. Near quantitative reduction of the Fe4S4 center by dithionite could be achieved if SiR was either converted to the CO complex or treated with 80% dimethyl sulfoxide. Spinach SiR and nitrite reductase (NiR) both catalyze Fdr-or MV+-de-pendent six-electron reductions of SO3(2)- and NO2-, as well as the two electron reduction of NH2OH. Vmax values are highest with the nitrogenous substrates. However, the Km of SiR for So3(2-), and of NiR for NO2-, is at least 2 orders of magnitude less than with either of the other substrates. Rates of reduction with Fdr as electron donor are greater than with MV+ as donor, No immunological cross-reaction could be detected between spinach SiR and Escherichia coli SiR or between spinach SiR and NiR.

Arabidopsis Proteins↗
PubMed2001

Structure of the electron transfer complex between ferredoxin and ferredoxin-NADP(+) reductase.

All oxygenic photosynthetically derived reducing equivalents are utilized by combinations of a single multifuctional electron carrier protein, ferredoxin (Fd), and several Fd-dependent oxidoreductases. We report the first crystal structure of the complex between maize leaf Fd and Fd-NADP(+) oxidoreductase (FNR). The redox centers in the complex--the 2Fe-2S cluster of Fd and flavin adenine dinucleotide (FAD) of FNR--are in close proximity; the shortest distance is 6.0 A. The intermolecular interactions in the complex are mainly electrostatic, occurring through salt bridges, and the interface near the prosthetic groups is hydrophobic. NMR experiments on the complex in solution confirmed the FNR recognition sites on Fd that are identified in the crystal structure. Interestingly, the structures of Fd and FNR in the complex and in the free state differ in several ways. For example, in the active site of FNR, Fd binding induces the formation of a new hydrogen bond between side chains of Glu 312 and Ser 96 of FNR. We propose that this type of molecular communication not only determines the optimal orientation of the two proteins for electron transfer, but also contributes to the modulation of the enzymatic properties of FNR.

Binding Sites↗
PubMed2001

Role of a cluster of hydrophobic residues near the FAD cofactor in Anabaena PCC 7119 ferredoxin-NADP+ reductase for optimal complex formation and electron transfer to ferredoxin.

In the ferredoxin-NADP(+) reductase (FNR)/ferredoxin (Fd) system, an aromatic amino acid residue on the surface of Anabaena Fd, Phe-65, has been shown to be essential for the electron transfer (ET) reaction. We have investigated further the role of hydrophobic interactions in complex stabilization and ET between these proteins by replacing three hydrophobic residues, Leu-76, Leu-78, and Val-136, situated on the FNR surface in the vicinity of its FAD cofactor. Whereas neither the ability of FNR to accept electrons from NADPH nor its structure appears to be affected by the introduced mutations, different behaviors with Fd are observed. Thus, the ET interaction with Fd is almost completely lost upon introduction of negatively charged side chains. In contrast, only subtle changes are observed upon conservative replacement. Introduction of Ser residues produces relatively sizable alterations of the FAD redox potential, which can explain the modified behavior of these mutants. The introduction of bulky aromatic side chains appears to produce rearrangements of the side chains at the FNR/Fd interaction surface. Thus, subtle changes in the hydrophobic patch influence the rates of ET to and from Fd by altering the binding constants and the FAD redox potentials, indicating that these residues are especially important in the binding and orientation of Fd for efficient ET. These results are consistent with the structure reported for the Anabaena FNR.Fd complex.

Amino Acid Sequence↗