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PubMed1999

A model for the solution structure of oxidized terpredoxin, a Fe2S2 ferredoxin from Pseudomonas.

Terpredoxin (Tdx) is a 105-residue bacterial ferredoxin consisting of a single polypeptide chain and a single Fe2S2 prosthetic group. Tdx was first identified in a strain of Pseudomonas sp. capable of using alpha-terpineol as sole carbon source. The Tdx gene, previously cloned from the plasmid-encoded terp operon, that carries genes encoding for proteins involved in terpineol catabolism, has been subcloned and expressed as the holoprotein in E. coli. Physical characterization of the expressed Tdx has been performed, and a model for the solution structure of oxidized Tdx (Tdxo) has been determined. High-resolution homo- and heteronuclear NMR data have been used for structure determination in diamagnetic regions of the protein. The structure of the metal binding site (which cannot be determined directly by NMR methods due to paramagnetic broadening of resonances) was modeled using restraints obtained from a crystal structure of the homologous ferredoxin adrenodoxin (Adx) and loose restraints determined from paramagnetic broadening patterns in NMR spectra. Essentially complete 1H and 15N NMR resonance assignments have been made for the diamagnetic region of Tdxo (ca. 80% of the protein). A large five-stranded beta-sheet and a smaller two-stranded beta-sheet were identified, along with three alpha-helices. A high degree of structural homology was observed between Tdx and two other ferredoxins with sequence and functional homology to Tdx for which structures have been determined, Adx and putidaredoxin (Pdx), a homologous Pseudomonas protein. 1H/2H exchange rates for Tdx backbone NH groups were measured for both oxidation states and are rationalized in the context of the Tdx structure. In particular, an argument is made for the importance of the residue following the third ligand of the metal cluster (Arg49 in Tdx, His49 in Pdx, His56 in Adx) in modulating protein dynamics as a function of oxidation state. Some differences between Tdx and Pdx are detected by UV-visible spectroscopy, and structural differences at the C-terminal region were also observed. Tdx exhibits only 2% of the activity of Pdx in turnover assays performed using the reconstituted camphor hydroxylase system of which Pdx is the natural component.

Amino Acid Sequence↗
PubMed1999

Effects of mutations in aspartate 14 on the spectroscopic properties of the [Fe3S4]+,0 clusters in Pyrococcus furiosus ferredoxin.

The properties of [Fe(3)S(4)](+,0) clusters in wild-type and mutant forms of Pf Fd with Asp, Ser, Cys, Val, His, Asn, and Tyr residues occupying position 14, i.e., proximal to the three micro(2)-S atoms of the cluster, have been investigated by the combination of EPR, variable-temperature magnetic circular dichroism (VTMCD), and resonance Raman (RR) spectroscopies. Two distinct types of [Fe(3)S(4)] clusters are identified on the basis of the breadth of the S = (1)/(2) [Fe(3)S(4)](+) EPR resonances and the marked differences in the VTMCD spectra of the S = 2 [Fe(3)S(4)](0) clusters. On the basis of the available NMR data for [Fe(3)S(4)](+, 0) clusters in ferredoxins, the distinctive properties of these two types of [Fe(3)S(4)] clusters are interpreted in terms of different locations of the more strongly coupled pair of irons in the oxidized clusters and the valence-delocalized pair in the reduced clusters. Near-IR VTMCD measurements indicate the presence of S = (9)/(2) valence-delocalized pairs in both types of [Fe(3)S(4)](0) clusters, and the spin-dependent delocalization energies associated with the Fe-Fe interactions were determined to be approximately 4300 cm(-)(1) in both cases. We conclude that the nature of the residue at position 14 in Pyrococcus furiosus ferredoxin is an important determinant of the location of the reducible pair of irons in a [Fe(3)S(4)](+,0) cluster, and the redox properties of the wild-type and mutant ferredoxins are discussed in light of these new results.

Aspartic Acid↗
PubMed2003

Theoretical investigation of the "CO in"-"CO out" isomerization in a [2Fe-2S] ferredoxin: free energy profiles and redox states.

This paper reports on extensive molecular dynamics simulations (about 40 ns in total) in both the reduced and the oxidized states of Ferredoxin from Cyanobacterium Anabaena PCC7119. These calculations have provided us with the free energy profile of the phi(47) backbone angle which controls the "CO in" to "CO out" transition of Cys46 in the reduced and oxidized Fd7119. Our main motivation has been to identify the time scales involved in the reduction of Fd and single out the amino acid residues crucially affecting the conformational change and, thus, electron transfer. The free energy profiles obtained in this study are relevant to electron transfers in the PSI/Fd7119 and Fd7119/FNR complexes. Our findings based on hydrated ferredoxin simulations are that activated processes are to occur in the protein during electron transfer to and from ferredoxin. The relative stability and the activation barrier of the "CO in" to "CO out" transition can be modulated by the distance between the Ser47 and the Glu94 residues. In our calculations, for short distances, the "CO in" state is favored in the reduced form, whereas for large distances, the "CO out" state becomes increasingly favored. Accordingly, conformational changes in Fd7119 when bound to PSI or FNR can have crucial effects on the kinetics of the electron transfer. Our simulations also show that the hydrogen bond between between Ser47(OG) and Cys46(O) is essential to lock in the "CO out" state. This finding explains why only the Ser47Thr Fd7119 mutant sustains electron transfer activity, as only residues serine and threonine can form a specific hydrogen bond with Cys46(O). Finally, our simulations predict that Phe65 has a large probability of being in close contact with the Cys46(O) at the top of the conformational free energy barrier. This carbonyl/phenyl ring interaction can then facilitate the de-localization of the Fd's electron toward the Pi orbitals of Phe65 aromatic ring which is thought to be crucial to the Fd7119/FNR electron transfer

Carbon Monoxide↗
PubMed2003

Ferredoxin from sweet pepper (Capsicum annuum L.) intensifying harpin(pss)-mediated hypersensitive response shows an enhanced production of active oxygen species (AOS).

The hypersensitive response (HR) is a form of cell death associated with plant resistance to pathogen infection. Harpin(pss), an elicitor from the bacterium Pseudomonas syringae pv. syringae, induces a HR in non-host plants. Previously, we reported an amphipathic protein from sweet pepper interfering with harpin(pss)-mediated HR. In this report, we isolated and characterized a cDNA clone encoded that amphipathic protein from sweet pepper. This protein is designated as PFLP (plant ferredoxin-like protein) by virtue of its high homology with plant ferredoxin protein containing an N-terminal signal peptide responsible for chloroplast targeting and a putative 2Fe-2S domain responsible for redox activity. Recombinant PFLP obtained from Escherichia coli was able to significantly increase active oxygen species (AOS) generation when mixed with harpin(pss) in tobacco suspension cells. It also showed enhanced HR when co-infiltrated with harpin(pss) in tobacco leaves. We used a transgenic tobacco suspension cells system that constitutively expresses the Pflp gene driven by the CaMV 35S promoter to study the function of PFLP in enhancing harpin(pss)-mediated hypersensitive cell death in vivo. In response to harpin(pss), suspension cells derived from Pflp transgenic tobacco showed a significant increase both in the generation of AOS and in cell death as compared to the wild type. AOS inhibitors diphenylene iodonium chloride (DPI) and lanthanum chlorate (LaCl3) were used to study the involvement of AOS in harpin(pss)-induced cell death. Our results demonstrate enhanced generation of AOS is necessary to cause enhanced hypersensitive cell death in Pflp transgenic tobacco cells and it is plasma membrane-bound NADPH-oxidase-dependent. Sub-cellular localization studies showed that PFLP is present in the cytoplasm and chloroplast of Pflp transgenic tobacco cells, but only in the chloroplast, not in the cytoplasm, of wild-type tobacco cells. It is possible that PFLP can change the redox state of the cell upon harpin(pss) inoculation to increase AOS generation and hypersensitive cell death. Overall, this study will provide a new insight in the functional properties of ferredoxin in hypersensitive cell death.

Amino Acid Sequence↗
PubMed1973

A four-iron ferredoxin from Desulfovibrio desulfuricans.

Ferredoxin from Desulfovibrio desulfuricans was isolated, purified and crystallized. It contains four iron atoms and four sulphido or ;acid-labile' sulphur atoms for a molecule of 6000 daltons. The absorption spectrum in the u.v.-visible region and the electron-paramagnetic-resonance signals of the reduced protein are similar to those observed for other four-iron ferredoxins. The amino acid composition is different from that of Desulfovibrio gigas ferredoxin. The redox potential of -0.33V at pH7.0 was determined by dye techniques.

Amino Acids↗
PubMed1979

Chloroplast cytochrome b-563, hydrophobic environment and lack of direct reaction with ferredoxin.

1. The reaction of electron donors with cytochrome b-563 in chloroplasts was studied by investigating their effects on the rate of its reduction in the presence of dithionite, which reacts only slowly with the cytochrome. The relative effects of 9,10-anthraquinone and 9,10-anthraquinone 2-sulphonate in the presence of dithionite suggested that the site of attack of redox reagents was protected behind a hydrophobic barrier from the external medium. 2. Ferredoxin had no measurable effect on the rate of reduction of the cytochrome in the presence of dithionite. 3. The reduction of pigment P700 in the dark after illumination in the presence of the inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea was stimulated by a combination of NADPH and ferredoxin, although NADPH alone had little effect. The same combination was unable to reduce cytochrome b-563 at a measurable rate. 4. It is concluded that the cytochrome is unlikely to be part of a linear pathway of electron flow between ferredoxin and pigment P700.

Anthraquinones↗
PubMed1987

Purification and characterization of pyruvate: ferredoxin oxidoreductase from the anaerobic protozoon Trichomonas vaginalis.

The pyruvate: ferredoxin oxidoreductase from the anaerobic protozoon Trichomonas vaginalis is an extrinsic protein bound to the hydrogenosomal membrane. It has been solubilized and purified to homogeneity, principally by salting-out chromatography on Sepharose 4B. Low recoveries of active enzyme were caused by inactivation by O2 and the irreversible loss of thiamin pyrophosphate. It is a dimeric enzyme of overall Mr 240,000 and subunit Mr 120,000. The enzyme contains, per mol of dimer, 7.3 +/- 0.3 mol of iron and 5.9 +/- 0.9 mol of acid-labile sulphur, suggesting the presence of two [4Fe-4S] centres, and 0.47 mol of thiamin pyrophosphate. The absorption spectrum of the enzyme is characteristic of a non-haem iron protein. The pyruvate: ferredoxin oxidoreductase from T. vaginalis is therefore broadly similar to the 2-oxo acid: ferredoxin (flavodoxin) oxidoreductases purified from bacterial sources, except that it is membrane-bound.

Amino Acids↗
PubMed1998

Voltammetric studies of the reactions of iron-sulphur clusters ([3Fe-4S] or [M3Fe-4S]) formed in Pyrococcus furiosus ferredoxin.

Reactions of the [3Fe-4S] cluster and various metallated [M3Fe-4S] adducts co-ordinated in the ferredoxin from the hyperthermophile Pyrococcus furiosus have been studied by protein-film voltammetry, bulk-solution voltammetry, solution kinetics and magnetic CD (MCD). The [3Fe-4S] cluster exhibits two couples, [3Fe-4S]+/0 and [3Fe-4S]0/2-. Film voltammetry is possible over a wide pH range (2-8), revealing that the [3Fe-4S]+/0 couple shows a complex pH dependence with pKred1=2.8, pKox=4.9 and pKred2=6.7. From MCD, pKred1 corresponds with protonation of [3Fe-4S]0 to give a spectroscopically distinct species, as reported for ferredoxins from Azotobacter and Sulfolobus. The status of the disulphide/disulphydryl entity makes no significant difference to the data (given for the -S-S- form). Formation of the hyper-reduced [3Fe-4S]2- state is observed, requiring 3H+ for the overall 3e- reduction of [3Fe-4S]+, the change therefore being electroneutral. By comparison with the ferredoxin from Desulfovibrio africanus, uptake of Fe(II) and other M(II) by [3Fe-4S]0 to give [M3Fe-4S] clusters is slow (t1/2>10 min at room temperature, slower still if the protein is adsorbed on the electrode), whereas reaction with Tl(I) to produce [Tl3Fe-4S] is very rapid (t1/2<<1 s), suggesting that co-ordination of Tl does not require reorganization of the protein structure. Rates of formation of [3Fe-4S] from [M3Fe-4S] adducts increase sharply at high potentials, showing that metal release involves a labile 'super-oxidized' [M3Fe-4S]3+ state.

Bacterial Proteins↗
PubMed1998

Targeted deletion and mutational analysis of the essential (2Fe-2S) plant-like ferredoxin in Synechocystis PCC6803 by plasmid shuffling.

The genes encoding (2Fe-2S) plant-like ferredoxins were studied in the widely used cyanobacterium Synechocystis PCC6803. The fedl gene (ssI0020) coding for the most abundant ferredoxin product was found to be expressed strongly as a light-induced monocistronic transcript, whereas the other fed genes appeared to be silent (sIr1828) or moderately expressed as polycistronic transcripts regulated by either light fluence (sIr0150, negative control) or glucose availability (sII1382). fedl was found to be critical to Synechocystis PCC6803 viability in spite of sIr0150, sII1382 or flavodoxin induction, even after the addition of glucose that compensates for the loss of photosynthesis. Nevertheless, fedl could be deleted from all chromosome copies in cells propagating a fedl gene (even of heterologous origin) on a replicating plasmid. This strain was used as the host for the subsequent introduction of fedl mutant alleles propagated on a second vector. Analysis of the fedl mutant strains generated after plasmid exchange showed that the C18-C85 disulphide bridge is not central either to the tight compaction of ferredoxin I or to its reduction by photosystem I and demonstrated that the length of the Fedl carboxy terminus is important for effective PSI/FedI interactions. The plasmid-shuffling strategy presently described has general applicability for mutational analysis of essential genes in many organisms, as it is based on promiscuous plasmids.

Bacterial Proteins↗
PubMed1993

Promoter and leader regions involved in the expression of the Arabidopsis ferredoxin A gene.

Ferredoxin is a nuclear-encoded protein that is involved in a variety of electron transfer reactions in both photosynthetic and non-photosynthetic plastids. We show here that the expression of the ferredoxin A gene (FedA) in Arabidopsis thaliana is light-regulated, with its mRNA level increased 4.5-fold by transfer of dark-grown seedlings to white light for 3 h. A portion of this light regulation is mediated by phytochrome through a very low fluence type of response. In addition, it is likely that another photoreceptor(s) is also involved. The FedA promoter confers a light- and tissue-regulated expression pattern when fused to the beta-glucuronidase (GUS) and luciferase reporter genes, indicating that the gene is transcriptionally regulated. No evidence of cis-acting light-regulatory elements within the 5' untranslated leader region of the gene was detected. Nevertheless, elements within this leader are required for full activity since its deletion reduces expression both in the light and dark by 25-fold. This region includes a sequence, ACAAAA, which is also present in the 5' untranslated leader of the other three ferredoxin leaders that have been sequenced and in the leaders of 31 other plant genes or about 8% of all plant genes in the GenBank database. In addition to mediating light regulation, the FedA promoter also directs GUS expression in aerial tissues at 70-fold higher levels than in roots. GUS activity staining in aerial tissues is observed in both photosynthetic and non-photosynthetic cells. These data indicate that the FedA promoter also carries the information for expression of this gene in a highly tissue- and cell-specific manner.

Arabidopsis↗
PubMed2002

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.

Anabaena↗
PubMed2000

Characterization of a nif-regulated flavoprotein (FprA) from Rhodobacter capsulatus. Redox properties and molecular interaction with a [2Fe-2S] ferredoxin.

A flavoprotein from Rhodobacter capsulatus was purified as a recombinant (His)6-tag fusion from an Escherichia coli clone over-expressing the fprA structural gene. The FprA protein is a homodimer containing one molecule of FMN per 48-kDa monomer. Reduction of the flavoprotein by dithionite showed biphasic kinetics, starting with a fast step of semiquinone (SQ) formation, and followed by a slow reduction of the SQ. This SQ was in the anionic form as shown by EPR and optical spectroscopies. Spectrophotometric titration gave a midpoint redox potential for the oxidized/SQ couple of Em1 = +20 mV (pH 8.0), whereas the SQ/hydroquinone couple could not be titrated due to the thermodynamic instability of SQ associated with its slow reduction process. The inability to detect the intermediate form, SQ, upon oxidative titration confirmed this instability and led to an estimate of Em2 - Em1 of > 80 mV. The reduction of SQ by dithionite was significantly accelerated when the [2Fe-2S] ferredoxin FdIV was used as redox mediator. The midpoint redox potential of this ferredoxin was determined to be -275 +/- 2 mV at pH 7.5, consistent with FdIV serving as electron donor to FprA in vivo. FdIV and FprA were found to cross-react when incubated together with the 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, giving a covalent complex with an Mr of approximately 60 000. Formation of this complex was unaffected by the redox states of the two proteins. Other [2Fe-2S] ferredoxins, including FdV and FdVI from R. capsulatus, were ineffective as electron carriers to FprA, and cross-reacted poorly with the flavoprotein. The possible function of FprA with regard to nitrogen fixation was investigated using an fprA-deleted mutant. Although nitrogenase activity was significantly reduced in the mutant compared with the wild-type strain, nitrogen fixation was apparently unaffected by the fprA deletion even under iron limitation or microaerobic conditions.

Amino Acid Sequence↗
PubMed1970

Proton magnetic resonance study of ferredoxin from Clostridium pasteurianum.

Magnetic susceptibilities of both reduced and oxidized ferredoxin from Clostridium pasteurianum were obtained in solution. Whereas the reduced form exhibits a Curie law behavior, the magnetic susceptibility of oxidized ferredoxin in fact increases with temperature and suggests extensive antiferromagnetic exchange coupling between the component iron atoms. Contact-shifted resonances are observed for both forms of ferredoxin that are attributed to the beta-CH(2) protons of the eight cysteine residues. A model based on these results is presented.

Clostridium↗
PubMed1971

Primary reactions of photosynthesis: photoreduction of a bound chloroplast ferredoxin at low temperature as detected by EPR spectroscopy.

An electron paramagnetic resonance signal was observed at 25 degrees K in whole spinach chloroplasts after illumination at 77 degrees K. The light-induced epr spectrum had g-values (g(x) = 1.86, g(y) = 1.94, g(z) = 2.05) and a temperature dependence that were characteristic of the reduced state of a plant-type ferredoxin. The light-induced epr spectrum was also observed in broken spinach chloroplasts from which soluble ferredoxin was removed. Chemical analyses showed that both whole and broken spinach chloroplasts contained amounts of nonheme iron and "acid-labile sulfide" consistent with the presence of a bound iron-sulfur protein, at a level of about one molecule per 75 chlorophyll molecules. These results support the conclusion that chloroplasts contain a bound ferredoxin that may serve as a primary low-potential electron acceptor in photosynthesis.

Chloroplasts↗
PubMed1971

Proton magnetic resonance studies of the ferredoxins from spinach and parsley.

Contact-shifted resonances have been detected in the pmr spectra of both oxidized and reduced forms of spinach and parsley ferredoxins. These resonances are assigned to the beta-CH(2) protons of four cysteine residues that are thought to bind the iron-sulfur redox center to the polypeptide chain. Temperature dependences of contact shifts reveal that the two iron atoms are antiferromagnetically coupled in both redox forms of each of these proteins. Thermal population of magnetic states gives rise to the contact shifts observed in the formally diamagnetic oxidized forms of these ferredoxins and accounts for the failure of contact shifts in the reduced forms exhibit to a Curie Law temperature dependence. It appears that the unpaired electron of reduced spinach and parsley ferredoxin is unequally distributed over the two iron centers. Valence states for the iron pairs of high-spin Fe(+3)-Fe(+3) and Fe(+2)-Fe(+3) for the oxidized and reduced forms, respectively, are compatible with the nmr results.

Electron Spin Resonance Spectroscopy↗
PubMed1972

A comparison of Fe 4 S 4 clusters in high-potential iron protein and in ferredoxin.

The structures of both oxidized (HP(ox)) and reduced (HP(red)) high-potential iron protein and of oxidized ferredoxin (Fd(ox)) have been partially refined at 2.0-A resolution by methods similar to those applied to the protein rubredoxin [Watenpaugh, K. D., Sieker, L. C., Herriott, J. R. & Jensen, L. H. (1971) Cold Spring Harbor Symp. Quant. Biol. 36, 359-367]. Average bond lengths and angles in the HP(red) and Fd(ox) Fe(4)S(4) (*) clusters are the same to within the root-mean square (rms) deviation of each mean value. A preliminary comparison of the two HiPIP oxidation states indicates that the HP(ox) cluster is geometrically similar to the HP(red) cluster, but that it is smaller by 0.1-0.2 A in certain dimensions. The HiPIP and ferredoxin cluster geometry is also nearly identical to that reported recently for a synthetic analog [Herskovitz, T., Averill, B. A., Holm, R. A., Ibers, J. A., Phillips, W. D. & Weither, J. F. (1972) Proc. Nat. Acad. Sci. USA 69, 2437-2441]. An apparent paradox presented by the large difference between the HiPIP and ferredoxin electrode potentials can be resolved by the assumption that the Fe(4)S(4) (*) cluster has not two but three oxidation states. The fully oxidized (HP(ox)) and fully reduced (Fd(red)) clusters are paramagnetic, and the intermediate state is spin-paired [Tsibris, J. C. M. and Woody, R. W. (1970) Coord. Chem. Rev. 5, 417-458]. This hypothesis is supported by structural and spectroscopic evidence that the "paired-spin" state exists in both HP(red) and Fd(ox).

Crystallography↗
PubMed2000

A mitochondrial ferredoxin is essential for biogenesis of cellular iron-sulfur proteins.

Iron-sulfur (Fe/S) cluster-containing proteins catalyze a number of electron transfer and metabolic reactions. The components and molecular mechanisms involved in the assembly of the Fe/S clusters have been identified only partially. In eukaryotes, mitochondria have been proposed to execute a crucial task in the generation of intramitochondrial and extramitochondrial Fe/S proteins. Herein, we identify the essential ferredoxin Yah1p of Saccharomyces cerevisiae mitochondria as a central component of the Fe/S protein biosynthesis machinery. Depletion of Yah1p by regulated gene expression resulted in a 30-fold accumulation of iron within mitochondria, similar to what has been reported for other components involved in Fe/S protein biogenesis. Yah1p was shown to be required for the assembly of Fe/S proteins both inside mitochondria and in the cytosol. Apparently, at least one of the steps of Fe/S cluster biogenesis within mitochondria requires reduction by ferredoxin. Our findings lend support to the idea of a primary function of mitochondria in the biosynthesis of Fe/S proteins outside the organelle. To our knowledge, Yah1p is the first member of the ferredoxin family for which a function in Fe/S cluster formation has been established. A similar role may be predicted for the bacterial homologs that are encoded within iron-sulfur cluster assembly (isc) operons of prokaryotes.

Adrenodoxin↗
PubMed1995

Azotobacter vinelandii NADPH:ferredoxin reductase cloning, sequencing, and overexpression.

Azotobacter vinelandii ferredoxin I (AvFdI) controls the expression of another protein that was originally designated Protein X. Recently we reported that Protein X is a NADPH-specific flavoprotein that binds specifically to FdI (Isas, J.M., and Burgess, B.K. (1994) J. Biol. Chem. 269, 19404-19409). The gene encoding this protein has now been cloned and sequenced. Protein X is 33% identical and has an overall 53% similarity with the fpr gene product from Escherichia coli that encodes NADPH:ferredoxin reductase. On the basis of this similarity and the similarity of the physical properties of the two proteins, we now designate Protein X as A. vinelandii NADPH:ferredoxin reductase and its gene as the fpr gene. The protein has been overexpressed in its native background in A. vinelandii by using the broad host range multicopy plasmid, pKT230. In addition to being regulated by FdI, the fpr gene product is overexpressed when A. vinelandii is grown under N2-fixing conditions even though the fpr gene is not preceded by a nif specific promoter. By analogy to what is known about fpr expression in E. coli, we propose that FdI may exert its regulatory effect on fpr by interacting with the SoxRS regulon.

Amino Acid Sequence↗