Redox potentials of algal and cyanobacterial flavodoxins.

The redox potentials of flavodoxins from the cyanobacteria Synechococcus PCC 6301 (formerly Anacystis nidulans) and Nostoc strain MAC, and from the red alga Chondrus crispus, were determined by potentiometric titration. For the oxidized-semiquinone interconversion the potentials at pH 7.0 of the three flavodoxins were between -210 and -235 mV, and these were pH-dependent over the range pH 6.9-8.2. For the semiquinone-reduced interconversion the potentials of the cyanobacterial flavodoxins were close to -414 mV, and that for the algal flavodoxin, -370 mV, is the highest reported in this group of flavoproteins.     

The amino acid sequence of the Azotobacter vinelandii flavodoxin.

The amino acid sequence of a group II flavodoxin, the $\text{Azotobacter vinelandii}$ flavodoxin has been determined. The FMN-redox protein was shown to exist as a single polypeptide chain and to contain 179 amino acids. Despite the rather low amino acid sequence homology with the other flavodoxins sequenced, it is concluded that sequences of the group I and group II flavodoxins are homologous. The major differences between the group I and group II flavodoxins appears to be a lengthening in the C-terminal region in the group II flavodoxins.     

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.     

Tertiary structure of oxidized flavodoxin from an eukaryotic red alga Chondrus crispus at 2.35-A resolution. Localization of charged residues and implication for interaction with electron transfer partners

The crystal structure of the oxidized form of a flavodoxin from an eukaryotic red alga, Chondrus crispus, has been determined by multiple isomorphous replacement and anomalous scattering methods. A model of the 173 residues and flavin mononucleotide (FMN) has been refined by a restrained least squares method to a crystallographic R-factor of 22.6% using 6236 reflections between 6.0 and 2.35 A with F greater than 3 sigma F. This molecule has a sheet consisting of five parallel beta-strands with two alpha-helices on one side of the sheet and three on the other side, and has a (beta alpha)5 structure. The molecule incorporates a substantial insertion in beta 5, as in Anacystis nidulans flavodoxin, which distinguishes these flavodoxins from the short-chain type. The isoalloxazine ring of FMN is sandwiched between the side chains of Trp-56 and Tyr-98, with its C-7 and C-8 methyl groups being exposed to solvent. The phosphate group of FMN is located at the N-terminal end of alpha 1, and forms extensive hydrogen bonds with the loop (T8-T13) between beta 1 and alpha 1 of the protein. Six of the total 11 lysine residues are clustered at the opposing face to the FMN-binding site, while about two-thirds of the total 35 acidic residues are located in the half of the molecule which includes the FMN-binding site. Such localization of charged residues produces a dipole within the molecule, which may be important in its recognition of the other proteins participating in electron transfer reactions.     

Structure of the oxidized long-chain flavodoxin from Anabaena 7120 at 2 A resolution.

The structure of the long-chain flavodoxin from the photosynthetic cyanobacterium Anabaena 7120 has been determined at 2 A resolution by the molecular replacement method using the atomic coordinates of the long-chain flavodoxin from Anacystis nidulans. The structure of a third long-chain flavodoxin from Chondrus crispus has recently been reported. Crystals of oxidized A. 7120 flavodoxin belong to the monoclinic space group P2(1) with a = 48.0, b = 32.0, c = 51.6 A, and beta = 92 degrees, and one molecule in the asymmetric unit. The 2 A intensity data were collected with oscillation films at the CHESS synchrotron source and processed to yield 9,795 independent intensities with Rmerg of 0.07. Of these, 8,493 reflections had I > 2 sigma and were used in the analysis. The model obtained by molecular replacement was initially refined by simulated annealing using the XPLOR program. Repeated refitting into omit maps and several rounds of conjugate gradient refinement led to an R-value of 0.185 for a model containing atoms for protein residues 2-169, flavin mononucleotide (FMN), and 104 solvent molecules. The FMN shows many interactions with the protein with the isoalloxazine ring, ribityl sugar, and the 5'-phosphate. The flavin ring has its pyrimidine end buried into the protein, and the functional dimethyl benzene edge is accessible to solvent. The FMN interactions in all three long-chain structures are similar except for the O4' of the ribityl chain, which interacts with the hydroxyl group of Thr 88 side chain in A. 7120, while with a water molecule in the other two. The phosphate group interacts with the atoms of the 9-15 loop as well as with NE1 of Trp 57. The N5 atom of flavin interacts with the amide NH of Ile 59 in A. 7120, whereas in A. nidulans it interacts with the amide NH of Val 59 in a similar manner. In C. crispus flavodoxin, N5 forms a hydrogen bond with the side chain hydroxyl group of the equivalent Thr 58. The hydrogen bond distances to the backbone NH groups in the first two flavodoxins are 3.6 A and 3.5 A, respectively, whereas in the third flavodoxin the distance is 3.1 A, close to the normal value. Even though the hydrogen bond distances are long in the first two cases, still they might have significant energy because their microenvironment in the protein is not accessible to solvent.(ABSTRACT TRUNCATED AT 400 WORDS)     

Isolation, cloning, mapping, and nucleotide sequencing of the gene encoding flavodoxin in Escherichia coli.

The flavodoxins constitute a highly conserved family of small, acidic electron transfer proteins with flavin mononucleotide prosthetic groups. They are found in prokaryotes and in red and green algae, where they provide electrons at low potentials for the reduction of nitrogen by nitrogenase, for the light-dependent reduction of NADP+ in photosynthesis, and for the reduction of sulfite. Proteins with the physical characteristics of flavodoxins have been implicated in the reductive activation of pyruvate formate-lyase and cobalamin-dependent methionine synthase in Escherichia coli. We have purified flavodoxin to homogeneity from E. coli, determined its N-terminal amino acid sequence, and used this sequence to construct a 64-fold degenerate oligonucleotide probe for the flavodoxin gene. Because the phenotype of a flavodoxin mutant is not known, we used this degenerate probe to screen the phages of the Kohara library and identified two phages, with inserts mapping at approximately 16 min, that hybridized to the probe. The flavodoxin gene, designated fldA, was subcloned from the DNA in the overlap region of these two clones. The deduced amino acid sequence, determined by nucleotide sequencing of the flavodoxin gene, shows strong homology with flavodoxins from nitrogen-fixing bacteria and cyanobacteria. The fldA gene maps at 15.9 min on the E. coli chromosome and is transcribed in a counterclockwise direction.     

Structure predictions and surface charge of nitrogenase flavodoxins from Klebsiella pneumoniae and Azotobacter vinelandii

A first approximation to the tertiary structure of the nitrogenase flavodoxins of Klebsiella pneumoniae and Azotobacter vinelandii can be obtained by superimposing their amino acid sequences upon the crystallographically determined structure of the long-chain flavodoxin from Anacystis nidulans. This procedure is validated by secondary structure predictions based on the sequence alone and by the distribution of polar and hydrophobic residues. It reveals, among other things, a distinctive distribution of surface charge peculiar to the nitrogenase flavodoxins, which is probably important in determining the kinetics of electron transfer with their physiological redox partners. The most likely positions of the phosphodiester bridge which has been described in the A. vinelandii molecule can also be assessed.     

Crystal structure of oxidized flavodoxin from a red alga Chondrus crispus refined at 1.8 A resolution. Description of the flavin mononucleotide binding site.

In order to describe the detailed conformation of the oxidized flavodoxin from a eukaryotic red alga, Chondrus crispus, the crystal structure has been refined by a restrained least-squares method. The crystallographic R factor is 0.168 for 13,899 reflections with F greater than 2 sigma F between 6.0 and 1.8 A resolution. The refined model includes 173 amino acid residues, flavin mononucleotide (FMN) and 110 water molecules. The root-mean-square deviation in bond lengths from ideal values is 0.015 A, and the mean co-ordinate error is estimated to be 0.2 A. The FMN is located at the periphery of the molecule. The orientation of the isoalloxazine ring is such that the C-7 and C-8 methyl groups are exposed to solvent and the pyrimidine moiety is buried in the protein. Three peptide segments, T8-T13, T55-T58 and D94-C103, are involved in FMN binding. The first segment of T8-T13 enfolds the phosphate group of the FMN. The three oxygen atoms in the phosphate group form extensive hydrogen bonds with amide groups of the main chain and the O gamma atoms of the side-chains in this segment. T55 O and W56 N epsilon 1 in the second segment form hydrogen bonds with O-2 in the ribityl moiety and one of the oxygen atoms in the phosphate group, respectively. The O gamma H of T58 forms a hydrogen bond with the N-5 atom in the isoalloxazine ring, which is expected to be protonated in the semiquinone form. The third segment is in contact with the isoalloxazine ring. It appears that the hydrogen bond acceptor of the NH of Asp94 in the third segment is O-2 rather than N-1 in the isoalloxazine ring. The isoalloxazine ring is flanked by the side-chains of Trp56 and Tyr98; it forms an angle of 38 degrees with the indole ring of Trp56 and is almost parallel to the benzene ring of Tyr98. The environment of the phosphate group is conserved as in other flavodoxins whereas that of the isoalloxazine ring differs. The relationship between the hydrogen bond to the N-5 in the ring and the redox potential for the oxidized/semiquinone couple is discussed.     

The evolution of protein sequences by repetitious gene duplication: Clostridial flavodoxin

Summary Internal regularities of amino acid sequences of flavodoxins, FMN-containing, low molecular weight flavoproteins, were statistically examined using the minimum mutation method. The sequence ofClostridium pasteurianum flavodoxin shows statistically significant evidence of repetitious internal gene duplications at different levels of structure. Peptide pairs with a low chance probability of occurrence were frequently observed at a shift of 5 residues. The pairs with the lowest chance probabilites are a pair of heptapeptides at positions 39–45 vs. 44–50, a 5 residue shift (p = 9 × 10^–6). Most of the related pairs are consistent and could best be explained by the repeating pentapeptide sequence: (Lys-Gly-Ala-Asp-Val-)_n and appropriate gaps. Internal repetitions with longer shifts were also suggested for other flavodoxins. Repetitious gene duplication is proposed for the early stages of flavodoxin evolution.     

Apoplastic oxidation of L-asparagine is involved in the control of the green algal endophyte Acrochaete operculata Correa & Nielsen by the red seaweed Chondrus crispus Stackhouse

Gametophytes of the marine alga Chondrus crispus are more resistant than tetrasporophytes to infection by the filamentous endophytic alga Acrochaete operculata. It has been shown recently that carrageenan oligosaccharides from the resistant gametophytic generation of C. crispus stimulate the secretion of L-asparagine (L-Asn) by the endophyte and that the host generates hydrogen peroxide and 2-oxo-succinamic acid after contact with this amino acid. Here the response of C. crispus to L-Asn and its effect on the pathogen is investigated. Chondrus crispus released hydrogen peroxide, ammonium ions, and a carbonyl compound into the medium when exposed to L-Asn. This response was correlated with an increase in oxygen consumption. Inhibitor studies indicated the involvement of a flavoenzyme in the reaction, which was sensitive to high concentrations of the reaction product, ammonium, and to chlorpromazine, quinacrine, and cyanide, inhibitors of L-amino acid oxidase. Cell wall macerate of C. crispus also responded to L-Asn, while protoplasts were inactive. Uptake of L-Asn into the cell was not necessary for the response, suggesting that the involved L-amino acid oxidase is apoplastic. Acrochaete operculata was more sensitive to hydrogen peroxide than C. crispus and settlement of A. operculata zoospores on C. crispus was reduced by 86% in the presence of L-Asn. This reduced settlement could be prevented with catalase. Chondrus crispus thus features an apoplastic amino acid oxidase, which is involved in the control of its endophytic pathogen. The modulation of the amino acid secretion in A. operculata by carrageenan oligosaccharides is therefore a key issue in the etiology of the association.     

Comparisons of wild-type and mutant flavodoxins from Anacystis nidulans. Structural determinants of the redox potentials

The long-chain flavodoxins, with 169-176 residues, display oxidation-reduction potentials at pH 7 that vary from -50 to -260 mV for the oxidized/semiquinone (ox/sq) equilibrium and are -400 mV or lower for the semiquinone/hydroquinone (sq/hq) equilibrium. To examine the effects of protein interactions and conformation changes on FMN potentials in the long-chain flavodoxin from Anacystis nidulans (Synechococcus PCC 7942), we have determined crystal structures for the semiquinone and hydroquinone forms of the wild-type protein and for the mutant Asn58Gly, and have measured redox potentials and FMN association constants. A peptide near the flavin ring, Asn58-Val59, reorients when the FMN is reduced to the semiquinone form and adopts a conformation ("O-up") in which O 58 hydrogen bonds to the flavin N(5)H; this rearrangement is analogous to changes observed in the flavodoxins from Clostridium beijerinckii and Desulfovibrio vulgaris. On further reduction to the hydroquinone state, the Asn58-Val59 peptide in crystalline wild-type A. nidulans flavodoxin rotates away from the flavin to the "O-down" position characteristic of the oxidized structure. This reversion to the conformation found in the oxidized state is unusual and has not been observed in other flavodoxins. The Asn58Gly mutation, at the site which undergoes conformation changes when FMN is reduced, was expected to stabilize the O-up conformation found in the semiquinone oxidation state. This mutation raises the ox/sq potential by 46 mV to -175 mV and lowers the sq/hq potential by 26 mV to -468 mV. In the hydroquinone form of the Asn58Gly mutant the C-O 58 remains up and hydrogen bonded to N(5)H, as in the fully reduced flavodoxins from C. beijerinckii and D. vulgaris. The redox and structural properties of A. nidulans flavodoxin and the Asn58Gly mutant confirm the importance of interactions made by N(5) or N(5)H in determining potentials, and are consistent with earlier conclusions that conformational energies contribute to the observed potentials.The mutations Asp90Asn and Asp100Asn were designed to probe the effects of electrostatic interactions on the potentials of protein-bound flavin. Replacement of acidic by neutral residues at positions 90 and 100 does not perturb the structure, but has a substantial effect on the sq/hq equilibrium. This potential is increased by 25-41 mV, showing that electrostatic interaction between acidic residues and the flavin decreases the potential for conversion of the neutral semiquinone to the anionic hydroquinone. The potentials and the effects of mutations in A. nidulans flavodoxin are rationalized using a thermodynamic scheme developed for C. beijerinckii flavodoxin.     

Structural and chemical properties of a flavodoxin from Anabaena PCC 7119

Structural and chemical properties of a flavodoxin from Anabaena PCC 7119 are described. The first 36 residues of the amino-terminal amino acid sequence have been determined and show extensive homology with flavodoxins isolated from other sources. Anabaena flavodoxin exhibits a net negative change (-3) in the helix-1 segment as found with other cyanobacterial flavodoxins Synechococcus 6301 (Anacystis nidulans) and Nostoc MAC, but in contrast to the net positive charge found in this region in the case of flavodoxins isolated from nitrogen-fixing bacteria (Azotobacter and Klebsiella). The FMN cofactor can be reversibly resolved from the apoprotein by trichloroacetic acid treatment. Apoflavodoxin, thus prepared, binds FMN with a Kd value of 0.1 nM and binds riboflavin with a decreased affinity (Kd = 5 microM) at pH 7.2. The apoprotein is stable in dilute solutions at pH values around 7 but readily denatures at pH 8 as judged from loss in flavin-binding ability and by ultraviolet circular dichroism spectroscopy. Oxidation-reduction potential studies at pH values of 7 and 8 show OX/SQ couples of -195 mV and -255 mV, respectively, and show SQ/HQ couples of -390 mV and -418 mV, respectively. From these data, the binding constant for the FMN semiquinone is calculated to be approx. 5-fold tighter and the binding of the FMN hydroquinone is approx. 10(5)-fold weaker than that of the oxidized FMN to the apoprotein. Anabaena flavodoxin functions as an effective mediator of electron transfer from ferredoxin-NADP(+)-reductase to cytochrome c with a turnover number [4.5-5) x 10(3) min-1); a values similar to that determined for Anabaena ferredoxin. The flavodoxin binds tightly to the reductase with Kd values of 6.4 and 8.5 microM at pH values of 7.0 and 8.0, respectively.     

Apoflavodoxin aggregation following dissociation of flavin

Flavodoxins were isolated for the cyanobacteria Anacystis nidulans and Nostoc strain MAC, and from the red alga Chondrus crispus, and apoflavodoxins prepared by five methods. Gel electrophoretic studies showed that whereas the apoproteins of A. nudulans and Nostoc strain MAC were recovered in monomeric form, the removal of riboflavin 5'-phosphate from C. crispus flavodoxin resulted in extensive aggregation of the apoprotein. In extent and nature this aggregation differed with the dissociating agent used.     

A flavodoxin that is required for enzyme activation: the structure of oxidized flavodoxin from Escherichia coli at 1.8 A resolution.

In Escherichia coli, flavodoxin is the physiological electron donor for the reductive activation of the enzymes pyruvate formate-lyase, anaerobic ribonucleotide reductase, and B12-dependent methionine synthase. As a basis for studies of the interactions of flavodoxin with methionine synthase, crystal structures of orthorhombic and trigonal forms of oxidized recombinant flavodoxin from E. coli have been determined. The orthorhombic form (space group P2(1)2(1)2(1), a = 126.4, b = 41.10, c = 69.15 A, with two molecules per asymmetric unit) was solved initially by molecular replacement at a resolution of 3.0 A, using coordinates from the structure of the flavodoxin from Synechococcus PCC 7942 (Anacystis nidulans). Data extending to 1.8-A resolution were collected at 140 K and the structure was refined to an Rwork of 0.196 and an Rfree of 0.250 for reflections with I > 0. The final model contains 3,224 non-hydrogen atoms per asymmetric unit, including 62 flavin mononucleotide (FMN) atoms, 354 water molecules, four calcium ions, four sodium ions, two chloride ions, and two Bis-Tris buffer molecules. The structure of the protein in the trigonal form (space group P312, a = 78.83, c = 52.07 A) was solved by molecular replacement using the coordinates from the orthorhombic structure, and was refined with all data from 10.0 to 2.6 A (R = 0.191; Rfree = 0.249). The sequence Tyr 58-Tyr 59, in a bend near the FMN, has so far been found only in the flavodoxins from E. coli and Haemophilus influenzae, and may be important in interactions of flavodoxin with its partners in activation reactions. The tyrosine residues in this bend are influenced by intermolecular contacts and adopt different orientations in the two crystal forms. Structural comparisons with flavodoxins from Synechococcus PCC 7942 and Anaebaena PCC 7120 suggest other residues that may also be critical for recognition by methionine synthase.     

Conformational changes in Chondrus crispus flavodoxin on dissociation of FMN and reconstitution with flavin analogues.

The apoflavodoxin produced by precipitation of Chondrus crispus flavodoxin with trichloroacetic acid migrates as a single molecular species on non-denaturing PAGE, but at a much lower Rm than the flavoprotein. Values of s and D were significantly lower than for the flavodoxin, but their substitution in the Svedberg equation indicated the molecular mass was closely similar to that of the flavodoxin. This was confirmed by meniscus-depletion sedimentation-equilibrium studies. The Stokes radius of the apoflavodoxin was 3.65 nm, compared with 2.33 nm for the flavodoxin, and estimates of frictional coefficient ratio suggested the apoprotein was in extended conformation compared with the roughly globular shape of the flavodoxin. The Ka for FMN binding was 2.8 x 10(8)M, and the electrophoretic and physicochemical properties of the reconstituted flavoprotein were closely similar to those of the native flavodoxin. FAD, iso-FMN and thio-FMN were also bound effectively, but methyl-FMN and riboflavin were bound only weakly, if at all. The reconstituted flavoproteins were active to various extents in mediating electron transfer from NADPH to cytochrome c catalysed by flavodoxin-NADP+ oxidoreductase, the highest activity being with the thio-FMN flavodoxin.     

Crystallization and preliminary X-ray diffraction studies of oxidized flavodoxin from Chondrus crispus, a red alga

Crystals of the oxidized form of flavodoxin from a red alga, Chondrus crispus, have been grown in ammonium sulfate solution by the dialysis method. The crystals belong to the orthorhombic system, space group P2(1)2(1)2(1), with unit cell dimensions of a = 63.6, b = 48.8, and c = 56.8 A. The asymmetric unit contains one molecule of flavodoxin. The crystals diffract X-rays to about 2.0 A resolution and are stable to X-ray beams. The diffraction patterns changed significantly upon soaking the crystal in a solution of a platinum complex. The major heavy-atom sites in the platinum derivative crystal have been identified from the difference Patterson function calculated at 4 A resolution.     

Proline inhibition of pyrroline-5-carboxylate reductase: differences in enzymes obtained from animal and tissue culture sources

The complete amino acid sequence for the 148 amino acid flavodoxin from $\text{Desulfovibrio}\text{vulgaris}$ is presented. This is the first flavoenzyme for which both the complete amino acid sequence and a 2.5 Å resolution x-ray diffraction structure are now known. The position of some important residues in the binding of FMN are given. The D. vulgaris sequence is compared with other published flavodoxin sequences.     

Cloning and characterization of the flavodoxin gene from Desulfovibrio desulfuricans

The gene coding for the flavodoxin protein from Desulfovibrio desulfuricans [Essex 6] (ATCC 29577) has been cloned and sequenced. The gene was identified on Southern blots of HindIII-digested genomic DNA by hybridization to the coding region for the flavodoxin from Desulfovibrio vulgaris [Hildenborough] (Krey, G.D., Vanin, E.F. and Swenson, R.P. (1988) J. Biol. Chem. 263, 15436-15443). Ultimately, a 1.8 kb TaqI fragment was cloned which contains an open reading frame of 447 nucleotides coding for an acidic protein of 148 amino acids and calculated molecular weight of 15,726. The derived amino acid sequence of this protein is 47% identical to the flavodoxin from D. vulgaris. Regions of the polypeptide which form the flavin mononucleotide binding site are largely homologous; however, some perhaps significant differences are noted. The aromatic amino acid residues that flank the flavin isoalloxazine ring in the D. vulgaris structure, i.e., tryptophan-60 and tyrosine-98, are conserved in this flavodoxin.     

The base sequence of the nifF gene of Klebsiella pneumoniae and homology of the predicted amino acid sequence of its protein product to other flavodoxins.

The nucleotide sequence of a 629 base-pair segment of DNA spanning the nifF gene of Klebsiella pneumoniae is presented. The structural gene comprises 531 base-pairs (175 codons, excluding the translational initiator and terminator) encoding an acidic polypeptide of 18950 Da. The nifF product thus belongs to the long-chain class of flavodoxins. It shows some sequence homology to the short-chain flavodoxins from Desulfovibrio vulgaris, Clostridium MP and Megasphaera elsdenii, and much stronger homology to long-chain flavodoxins from Azotobacter vinelandii and Anacystis nidulans. The long chain flavodoxins thus seem to constitute a well-conserved sub-group. The homology with the A. vinelandii flavodoxin is particularly strong, which may reflect their common function in nitrogen fixation.