Laser-flash absorption spectroscopy study of the competition between ferredoxin and flavodoxin photoreduction by Photosystem I in Synechococcus sp. PCC 7002: Evidence for a strong preference for ferredoxin

The competition between ferredoxin and flavodoxin for electrons from Photosystem I was analyzed by flash absorption spectroscopy of the photoreduction processes that take place in the presence of both acceptor proteins in vitro. Steady state photoreduction assays indicate a strong inhibition of the apparent flavodoxin photoreduction activities of Photosystem I in the presence of ferredoxin. Flash-absorption experiments carried out at 626 nm, a wavelength where the reduction of ferredoxin shows no spectral contribution, show that the photoreduction of oxidized flavodoxin and flavodoxin semiquinone are inhibited by ferredoxin in a quantitatively similar way. The experimental data can be satisfactorily described by a reaction model that assumes that both redox states of flavodoxin do not compete with ferredoxin for binding on PS I and that the binding equilibrium between ferredoxin and PS I is not changed in their presence. In contrast, a model which assumes that ferredoxin and flavodoxin actually compete for binding to PS I gives poor results. Similarly, experimental data observed in the presence of both redox states of flavodoxin can also be quantitatively described under the assumption that the binding equilibrium between flavodoxin semiquinone and PS I is not disturbed by oxidized flavodoxin. Taken together, this analysis shows that PS I favors ferredoxin over flavodoxin and flavodoxin semiquinone over oxidized flavodoxin. This behavior is in accordance with the values of the dissociation constants for complexes between PS I and its acceptors. However, in case of ferredoxin the observed preference is stronger than expected from these values, indicating that ferredoxin is almost absolutely preferred by PS I over flavodoxin and is always reduced first.     

Azotobacter vinelandii flavodoxin: purification and properties of the recombinant, dephospho form expressed in Escherichia coli

The nifF gene coding for the flavodoxin from the nitrogen-fixing bacterium Azotobacter vinelandii (strain OP) was cloned into the plasmid vector pUC7 [Bennett, L. T., Jacobsen, M. R., & Dean, D. R. (1988) J. Biol. Chem. 263 1364-1369] and the resulting plasmid transformed and expressed in Escherichia coli strain DH5. Recombinant Azotobacter flavodoxin is expressed at levels 5-6-fold higher in E. coli than in comparable yields of Azotobacter cultures grown under nitrogen-fixing conditions. Even higher levels were observed with flavodoxin expressed in E. coli under control of a tac promoter. Electron spin resonance spectroscopy on whole cells and in cell-free extracts showed the flavodoxin to be largely in the semiquinone form. The flavodoxin purified from E. coli exhibited the same molecular weight, isoelectric point, flavin mononucleotide (FMN) content, N-terminal sequence, and carboxyl-terminal amino acids as for the wild-type Azotobacter protein. The recombinant flavodoxin differed from native flavodoxin in that it exhibited an increased antigenicity to flavodoxin antibody and did not contain a covalently bound phosphate. Small differences are also observed in circular dichroism spectral properties in the visible and ultraviolet spectral regions. The recombinant, dephospho flavodoxin exhibits an oxidized/semiquinone potential (pH 8.0) of -224 mV and a semiquinone/hydroquinone couple (pH 8.0) of -458 mV. This latter couple is 50-60 mV higher than that exhibited by the native flavodoxin. Resolution of recombinant dephospho flavodoxin resulted in an apoflavodoxin that was much less stable than that prepared from the native protein.(ABSTRACT TRUNCATED AT 250 WORDS)     

A modified flavodoxin with altered redox potentials is less efficient in electron transfer to nitrogenase

The flavodoxins of the Azotobacter vinelandii wild-type and a mutant strain TZN 200 have been studied. Although the primary structure of the two proteins is the same, the ability of the mutant flavodoxin to donate electrons to nitrogenase is reduced by 75%. One reason may be the raised mid-point potential of -435 mV for the semiquinone/hydroquinone couple in the mutant flavodoxin. The respective redox potential for the wild-type flavodoxin was found to be -480 mV. As shown by paper chromatography and light absorption spectroscopy, the structure of FMN is modified in the TZN 200 flavodoxin.     

fldA is an essential gene required in the 2-C-methyl-D-erythritol 4-phosphate pathway for isoprenoid biosynthesis

Although flavodoxin I is indispensable for Escherichia coli growth, the exact pathway(s) where flavodoxin I is essential has not been identified. We performed transposon mutagenesis of the flavodoxin I gene, fldA, in an E. coli strain that expressed mevalonate pathway enzymes and that had a point mutation in the lytB gene of the MEP pathway resulting in the accumulation of (E)-4-hydroxy-3-methylbutyl-2-enyl pyrophosphate (HMBPP). Disruption of fldA abrogated mevalonate-independent growth and dramatically decreased HMBPP levels. The fldA- mutant grew with mevalonate indicating that the essential role of flavodoxin I under aerobic conditions is in the MEP pathway. Growth was restored by fldA complementation. Since GcpE (which synthesizes HMBPP) and LytB are iron-sulfur enzymes that require a reducing system for their activity, we propose that flavodoxin is essential for GcpE and possibly LytB activity. Thus, the essential role for flavodoxin I in E. coli is in the MEP pathway for isoprenoid biosynthesis.     

Ferredoxin and flavodoxin reduction by photosystem I.

Ferredoxin and flavodoxin are soluble proteins which are reduced by the terminal electron acceptors of photosystem I. The kinetics of ferredoxin (flavodoxin) photoreduction are discussed in detail, together with the last steps of intramolecular photosystem I electron transfer which precede ferredoxin (flavodoxin) reduction. The present knowledge concerning the photosystem I docking site for ferredoxin and flavodoxin is described in the second part of the review.     

Crystal structure of oxidized flavodoxin, an essential protein in Helicobacter pylori

The redox protein flavodoxin has been shown earlier to be reduced by the pyruvate-oxidoreductase (POR) enzyme complex of Helicobacter pylori, and also was proposed to be involved in the pathogenesis of gastric mucosa-associated lymphoid-tissue lymphoma (MALToma). Here, we report its X-ray structure, which is similar to flavodoxins of other bacteria and cyanobacteria. However, H. pylori flavodoxin has an alanine residue near the isoalloxazine ring of its cofactor flavin mononucleotide (FMN), while the other previously crystallized flavodoxins have a larger hydrophobic residue at this position. This creates a solute filled hole near the FMN cofactor of H. pylori flavodoxin. We also show that flavodoxin is essential for the survival of H. pylori, and conclude that its structure can be used as a starting point for the modeling of an inhibitor for the interaction between the POR-enzyme complex and flavodoxin.     

A new,fast, and sensitive assay for NADH-ferredoxin oxidoreductase detection in clostridia

A new, simple and very sensitive assay for NADH-ferredoxin or flavodoxin reductase activity is described. The assay is based on the nonenzymatic reduction of the metronidazole by ferredoxin or flavodoxin. In the presence of NADH, ferredoxin or flavodoxin and cell-free extract of clostridia, no metronidazole reduction is observed; the reaction occurs only if acetyl-CoA is added to the reaction mixture. Metronidazole reduction is quantitated by the spectrophotometric measurement at 320 nm. In this assay the change in absorbance is linearly related to the amount of clostridial extract for concentration of 0.1 to 0.8 mg/ml and to the flavodoxin or ferredoxin for concentrations of 0.5 to 8 nmol/ml.     

Redox potential difference between Desulfovibrio vulgaris and Clostridium beijerinckii flavodoxins

The redox potential of the flavin mononucleotide (FMN) hydroquinones for one-electron reduction in the Desulfovibrio vulgaris ( D. vulgaris) flavodoxin ( E sq/hq for FMNH (*)/FMNH (-)) was calculated using the crystal structure of the relevant hydroquinone form and compared to the results of the Clostridium beijerinckii ( C. beijerinckii) flavodoxin. In D. vulgaris and C. beijerinckii flavodoxins, the protein side chain causes significant downshifts of 170 and 240 mV in E sq/hq, respectively. In the C. beijerinckii flavodoxin, the E sq/hq downshift because of the protein side chain is essentially compensated by the counter influence of the protein backbone ( E sq/hq upshift of 260 mV). However, in the D. vulgaris flavodoxin, the corresponding protein backbone influence on E sq/hq is significantly small, i.e., less than half of that in the C. beijerinckii flavodoxin. In particular, there is a significant difference in the influence of the protein backbone of the so-called 60s loop region between the two flavodoxins. The E sq/hq difference can be best explained by the lower compensation of the side chain influence by the backbone influence in the D. vulgaris flavodoxin than in the C. beijerinckii flavodoxin.     

Structural alignment of ferredoxin and flavodoxin based on electrostatic potentials: implications for their interactions with photosystem I and ferredoxin-NADP reductase

The two proteins ferredoxin and flavodoxin can replace each other in the photosynthetic electron transfer chain of cyanobacteria and algae. However, structure, size, and composition of ferredoxin and flavodoxin are completely different. Ferredoxin is a small iron-sulfur protein (approximately 100 amino acids), whereas flavodoxin is a flavin-containing protein (approximately 170 amino acids). The crystal structure of both proteins from the cyanobacteria Anabeana PCC 7120 is known. We used these two protein structures to investigate the structural basis of their functional equivalence. We apply the Hodgkin index to quantify the similarity of their electrostatic potentials. The technique has been applied successfully in indirect drug design for the alignment of small molecule and bioisosterism elucidation. It requires no predefined atom-atom correspondences. As is known from experiments, electrostatic interactions are most important for the association of ferredoxin and flavodoxin with their reaction partners photosystem I and ferredoxin-NADP reductase. Therefore, use of electrostatic potentials for the structural alignment is well justified. Our extensive search of the alignment space reveals two alignments with a high degree of similarity in the electrostatic potential. In both alignments, ferredoxin overlaps completely with flavodoxin. The active sites of ferredoxin and flavodoxin rather than their centers of mass coincide in both alignments. This is in agreement with electron microscopy investigations on photosystem I cross-linked to ferredoxin or flavodoxin. We identify residues that may have the same function in both proteins and relate our results to previous experimental data.     

A pulse-radiolysis study of the reduction of flavodoxin from Megasphaera elsdenii by viologen radicals. A conformational change as a possible regulating mechanism

Pulse-radiolysis experiments were performed on solutions containing methyl or benzyl viologen and flavodoxin. Viologen radicals are formed after the pulse. The kinetics of the reaction of these radicals with flavodoxin were studied. The kinetics observed depend strongly on the concentration of oxidized viologen. Therefore one must conclude that a relatively stable intermediate is formed after the reduction of flavodoxin. The midpoint potential of the intermediate state is -(480 +/- 30) mV, and is hardly dependent on the pH between 7 and 9.2. Due to a conformational change (k2 approximately equal to 10(5)S-1) the intermediate state decays to the stable semiquinone form of flavodoxin. The delta G of the conformational change at pH 8 is about 29 kJ mol -1 (0.3 eV). This means that the upper limit for the pK of N-5 in the semiquinone form will be 13. The activation energy of the conformational change is 43 kJ mol -1 (0.45 eV). The reaction between methyl viologen radicals and the semiquinone of flavodoxin can be described by a normal bimolecular reaction. The reaction is diffusion-controlled with a forward rate constant of (7 +/- 1) X 10(8) M -1S -1 (pH 8, I = 55 mM). The midpoint potential of the semiquinone/hydroquinone was found to be -(408 +/- 5) mV. A consequence of the intermediate state is that flavodoxin (Fld) could be reduced by a two-electron process, the midpoint potential of which should be located between -440 mV less than Em (Fld/FldH-) less than -290 mV. The exact value will depend on the delta G of the conformational change between the fully reduced flavodoxin with its structure in the oxidized form and the fully reduced flavodoxin with its structure in the hydroquinone form. The conditions are discussed under which flavodoxin could behave as a two-electron donor.     

Physicochemical properties of flavodoxin from Desulfovibrio vulgaris.

Reductive titration curves of flavodoxin from Desulfovibrio vulgaris displayed two one-electron steps. The redox potential E-2 for the couple oxidized flavodoxin/flavodoxin semiquinone was determined by direct titration with dithionite. E-2 was -149 plus or minus 3 mV (pH 7.78, 25 degrees C). The redox potential E-1 for the couple flavodoxin semiquinone/fully reduced flavodoxin was deduced from the equilibrium concentration of these species in the presence of hydrogenase and H-2. E-1 was -438 plus or minus 8 mV (pH 7.78, 25 degrees C). Light-absorption and fluorescence spectra of flavodoxin in its three redox states have been recorded. Both the rate and extent of reduction of flavodoxin semiguinone with dithionite were found to depend on pH. An equilibrium between the semiquinone and hydroquinone forms occurred at pH values close to the neutrality, even in the presence of a large excess of dithionite, suggesting an ionization in fully reduced flavodoxin with a pK-a = 6.6. The association constants K for the three FMN redox forms with the apoprotein were deduced from the value of K (K = 8 times 10-7 M-1) measured with oxidized EMN at pH 7.0. Oxidized flavodoxin was found to comproportionate with the fully reduced protein (k-comp = 4.3 times 10-3 M-1 times s-1, pH 9.0, 22 degrees C) and with reduced free FMN (K-comp = 44 M-1 times s-1, pH 8.1, 20 degrees C). Fast oxidation of reduced flavodoxin occurred in the presence of O-2. Slower oxidation of semiquinone was dependent on pH in a drastic way.     

Consequences of the iron-dependent formation of ferredoxin and flavodoxin on photosynthesis and nitrogen fixation on Anabaena strains

Iron-dependent formation of ferredoxin and flavodoxin was determined in Anabaena ATCC 29413 and ATCC 29211 by a FPLC procedure. In the first species ferredoxin is replaced by flavodoxin at low iron levels in the vegetative cells only. In the heterocysts from Anabaena ATCC 29151, however, flavodoxin is constitutively formed regardless of the iron supply.Replacement of ferredoxin by flavodoxin had no effect on photosynthetic electron transport, whereas nitrogen fixation was decreased under low iron conditions. As ferredoxin and flavodoxin exhibited the same K_m values as electron donors to nitrogenase, an iron-limited synthesis of active nitrogenase was assumed as the reason for inhibited nitrogen fixation. Anabaena ATCC 29211 generally lacks the potential to synthesize flavodoxin. Under iron-starvation conditions, ferredoxin synthesis is limited, with a negative effect on photosynthetic oxygen evolution.     

Interaction of flavodoxin with cobalamin-dependent methionine synthase

Cobalamin-dependent methionine synthase catalyzes the transfer of a methyl group from methyltetrahydrofolate to homocysteine, forming tetrahydrofolate and methionine. The Escherichia coli enzyme, like its mammalian homologue, is occasionally inactivated by oxidation of the cofactor to cob(II)alamin. To return to the catalytic cycle, the cob(II)alamin forms of both the bacterial and mammalian enzymes must be reductively remethylated. Reduced flavodoxin donates an electron for this reaction in E. coli, and S-adenosylmethionine serves as the methyl donor. In humans, the electron is thought to be provided by methionine synthase reductase, a protein containing a domain with a significant degree of homology to flavodoxin. Because of this homology, studies of the interactions between E. coli flavodoxin and methionine synthase provide a model for the mammalian system. To characterize the binding interface between E. coli flavodoxin and methionine synthase, we have employed site-directed mutagenesis and chemical cross-linking using carbodiimide and N-hydroxysuccinimide. Glutamate 61 of flavodoxin is identified as a cross-linked residue, and lysine 959 of the C-terminal activation domain of methionine synthase is assigned as its partner. The mutation of lysine 959 to threonine results in a diminished level of cross-linking, but has only a small effect on the affinity of methionine synthase for flavodoxin. Identification of these cross-linked residues provides evidence in support of a docking model that will be useful in predicting the effects of mutations observed in mammalian homologues of E. coli flavodoxin and methionine synthase.     

Towards a new therapeutic target: Helicobacter pylori flavodoxin

Helicobacter pylori flavodoxin is the electronic acceptor of the pyruvate-oxidoreductase complex (POR) that catalyzes pyruvate oxidative decarboxilation. Inactivation of this metabolic route precludes bacterial survival. Because flavodoxin is not present in the human host, substances interfering electronic transport from POR might be well suited for eradication therapies against the bacterium. H. pylori flavodoxin presents a peculiar cofactor (FMN) binding site, compared to other known flavodoxins, where a conserved aromatic residue is replaced by alanine. A cavity thus appears under the cofactor that can be filled with small organic molecules. We have cloned H. pylori fldA gene, expressed the protein in Escherichia coli and characterized the purified flavodoxin. Thermal up-shift assays of flavodoxin with different concentrations of benzylamine, as well as fluorescence titration experiments indicate benzylamine binds in the pocket near the FMN binding site. It seems thus that low affinity inhibitors of H. pylori flavodoxin can be easily found that, after improvement, may give rise to leads.     

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.     

MioC is an FMN-binding protein that is essential for Escherichia coli biotin synthase activity in vitro

Biotin synthase is required for the conversion of dethiobiotin to biotin and requires a number of accessory proteins and small molecule cofactors for activity in vitro. We have previously identified two of these proteins as flavodoxin and ferredoxin (flavodoxin) NADP(+) reductase. We now report the identification of MioC as a third essential protein, together with its cloning, purification, and characterization. Purified MioC has a UV-visible spectrum characteristic of a flavoprotein and contains flavin mononucleotide. The presence of flavin mononucleotide and the primary sequence similarity to flavodoxin suggest that MioC may function as an electron transport protein. The role of MioC in the biotin synthase reaction is discussed, and the structure and function of MioC is compared with that of flavodoxin.     

Nitrogen fixation persists under conditions of salt stress in transgenic Medicago truncatula plants expressing a cyanobacterial flavodoxin

Several recent studies have demonstrated that the expression of a cyanobacterial flavodoxin in plants can provide tolerance to a wide range of environmental stresses. Indeed, this strategy has been proposed as a potentially powerful biotechnological tool to generate multiple‐tolerant crops. To determine whether flavodoxin expression specifically increased tolerance to salt stress and whether it might also preserve legume nitrogen fixation under saline conditions, the flavodoxin gene was introduced into the model legume Medicago truncatula. Expression of flavodoxin did not confer saline tolerance to the whole plant, although the sensitive nitrogen‐fixing activity was maintained under salt stress in flavodoxin‐expressing plants. Our results indicate that flavodoxin induced small but significant changes in the enzymatic activities involved in the nodule redox balance that might be responsible for the positive effect on nitrogen fixation. Expression of flavodoxin can be regarded as a potential tool to improve legume symbiotic performance under salt stress, and possibly other environmental stresses.     

Cloning, nucleotide sequence, and expression of the flavodoxin gene from Desulfovibrio vulgaris (Hildenborough)

The gene coding for the flavodoxin protein from Desulfovibrio vulgaris (Hildenborough) has been identified, cloned, and sequenced. DNA fragments containing the flavodoxin gene were identified by hybridization of a mixed synthetic heptadecanucleotide probe to Southern blots of SalI-digested genomic DNA. The nucleotide sequences of the probe were derived from the published protein primary structure (Dubourdieu, M., LeGall, J., and Fox, J. L. (1973) Biochem. Biophys. Res. Commun. 52, 1418-1425). The same oligonucleotide probe was used to screen libraries (in pUC19) containing size-selected SalI fragments. One recombinant, carrying a 1.6-kilobase (kb) insert which strongly hybridizes to the probe, was found to contain a nucleotide sequence which codes for the first 104 residues of the amino-terminal portion of the flavodoxin protein sequence but lacked the remainder of the gene. Therefore, a PstI restriction fragment from this clone was used as a probe to isolate the entire gene from a partial Sau3AI library in Charon 35. Of the plaques which continued to hybridize strongly to this probe through repeated screenings, one recombinant, containing a 16-kb insert, was further characterized. The entire flavodoxin gene was localized within a 1.4-kb XhoI-SacI fragment of this clone. The complete nucleotide sequence of the structural gene for the flavodoxin protein from Desulfovibrio vulgaris and flanking sequences which may include promoter and regulatory sequences are reported here. The cloned flavodoxin gene was placed behind the hybrid tac promoter for overexpression of the protein in Escherichia coli. Modification to the 5'-end of the gene, including substitutions at the second codon, were required to obtain high levels of expression. The expressed recombinant flavodoxin protein is isolated from E. coli cells as the holoprotein with physical and spectral properties similar to the protein isolated from D. vulgaris. To our knowledge, this is the first example of the expression of a foreign flavodoxin gene in E. coli using recombinant DNA methods.     

Purification and properties of a nif-specific flavodoxin from the photosynthetic bacterium Rhodobacter capsulatus.

A flavodoxin was isolated from iron-sufficient, nitrogen-limited cultures of the photosynthetic bacterium Rhodobacter capsulatus. Its molecular properties, molecular weight, UV-visible absorption spectrum, and amino acid composition suggest that it is similar to the nif-specific flavodoxin, NifF, of Klebsiella pneumoniae. The results of immunoblotting showed that R. capsulatus flavodoxin is nif specific, since it is absent from ammonia-replete cultures and is not synthesized by the mutant strain J61, which lacks a nif-specific regulator (NifR1). Growth of cultures under iron-deficient conditions causes a small amount of flavodoxin to be synthesized under ammonia-replete conditions and increases its synthesis under N2-fixing conditions, suggesting that its synthesis is under a dual system of control with respect to iron and fixed nitrogen availability. Here we show that flavodoxin, when supplemented with catalytic amounts of methyl viologen, is capable of efficiently reducing nitrogenase in an illuminated chloroplast system. Thus, this nif-specific flavodoxin is a potential in vivo electron carrier to nitrogenase; however, its role in the nitrogen fixation process remains to be established.