Essentiality of Boron for Dinitrogen Fixation in Anabaena sp. PCC 7119

The relationship between the requirement for boron and the form of N supplied in nutrient media to cyanobacterium Anabaena sp. PCC 7119 was investigated. When cells were grown in a medium which contained nitrate or ammonium-N, boron deficiency in the nutrient media did not inhibit growth or change cell composition. However, when cells were dependent on N₂ fixation, the lack of boron inhibited growth (i.e. growth ceased after 96 hours under these conditions). Additionally, boron-deficient cells showed a significant decrease in their content of phycobiliproteins and chlorophyll and accumulated carbohydrates within 24 hours of removing boron from the nutrient media. Inhibition of photosynthetic O₂ evolution accompanied the decrease in photosynthetic pigments. Boron deficiency symptoms were relieved when either boron or combined N was added to boron-deficient cultures. The degree of recovery depended upon the age of the cultures. Assays of nitrogenase activity showed that, after 2 hours of growth, nitrogenase activity of boron-deficient cells was inhibited by 40%. After 24 hours a total inactivation of nitrogenase activity was observed in boron-deficient cells. These results strongly suggest an involvement of boron in N₂ fixation in cyanobacteria.     

Boron Protection for O(2) Diffusion in Heterocysts of Anabaena sp. PCC 7119

The effect of boron on nitrogenase activity has been studied. When cells were dependent on N₂ fixation, the lack of boron inhibited nitrogenase activity. However, under anaerobic conditions or in the presence of Na-dithionite this effect was not observed. Nitrogenase synthesis was not affected by boron deficiency. Similarly, the heterocyst number was not altered. Examination of boron-deficient cultures showed, however, some dramatic changes in heterocyst morphology. The increased activity of those enzymes related to the maintaining of the low intracellular level of toxic oxygen species (superoxide dismutase, catalase, and peroxidase) support our hypothesis of the role of boron in heterocyst envelope stabilization.     

Functional domains of theEscherichia coli ferric uptake regulator protein (Fur)

The functions of N- and C-terminal domains of the Fur repressor ofEscherichia coli in promoter recognition and dimerization were studied. We investigated the ability of fusion proteins containing the N- or C-terminal domain of Fur to dimerize and to repress a Fur-regulatedlacZ fusion gene. The N-terminal domain, when fused to the C-terminal domain of the repressor C1857, repressed a Fur-regulatedlacZ fusion. However, the Fur-CI857 fusion was unable to complement the growth defect of anE. coli fur mutant on fumarate and succinate. The C-terminal domain of Fur, when fused to the N-terminus of CI857, repressed a λP, -regulatedlacZ fusion, indicating dimerization of the chimeric protein, which is a prerequisite for Cl activity. Both fusion proteins were fully active under both iron-rich and iron-poor growth conditions. We conclude that the N-terminal domain of Fur is involved in recognition of the Fur-responsive promoter and the C-terminus mediates oligomerization of the repressor.     

Functional domains of theEscherichia coli ferric uptake regulator protein (Fur)

The functions of N- and C-terminal domains of the Fur repressor ofEscherichia coli in promoter recognition and dimerization were studied. We investigated the ability of fusion proteins containing the N- or C-terminal domain of Fur to dimerize and to repress a Fur-regulatedlacZ fusion gene. The N-terminal domain, when fused to the C-terminal domain of the repressor C1857, repressed a Fur-regulatedlacZ fusion. However, the Fur-CI857 fusion was unable to complement the growth defect of anE. coli fur mutant on fumarate and succinate. The C-terminal domain of Fur, when fused to the N-terminus of CI857, repressed a P, -regulatedlacZ fusion, indicating dimerization of the chimeric protein, which is a prerequisite for Cl activity. Both fusion proteins were fully active under both iron-rich and iron-poor growth conditions. We conclude that the N-terminal domain of Fur is involved in recognition of the Fur-responsive promoter and the C-terminus mediates oligomerization of the repressor.     

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.     

Recognition of DNA by three ferric uptake regulator (Fur) homologs in Bacillus subtilis

Bacillus subtilis contains three Fur homologs: Fur, PerR, and Zur. Despite significant sequence similarities, they respond to different stimuli and regulate different sets of genes. DNA target site comparisons indicate that all three paralogs recognize operators with a core 7-1-7 inverted repeat. The corresponding consensus sequences are identical at five or more of the seven defined positions. Using site-directed mutagenesis, the Per box at the mrgA promoter was altered to mimic the core 7-1-7 motif of the Fur and Zur boxes. In vitro, the mrgA promoter containing a Zur box was only recognized by Zur, as demonstrated by DNase I footprinting assays. In contrast, both Fur and PerR bound to the mrgA promoter region containing a consensus Fur box. Expression analysis of these promoters is consistent with the in vitro data demonstrating as few as 1 or 2 base changes per half-site are sufficient to alter regulation. Similarly, the Fur box at the feuA promoter can be converted into a Per or a Zur box by appropriate mutations. While both Fur and PerR could recognize some of the same synthetic operator sequences, no naturally occurring sites are known that are subject to dual regulation. However, the PerR-regulated zosA gene is controlled from a regulatory region that contains both Per and Fur boxes. Although purified Fur protein bound to the candidate Fur boxes, Fur has little effect on zosA expression-possibly due to the location of the Fur boxes relative to the zosA promoter. Together, our results identify two nucleotide positions that are important for the ability of PerR, Fur, and Zur to distinguish among the many closely related operator sites present in the B. subtilis genome.     

Effects of the Alkaloid Gramine on the Light-Harvesting,Energy Transfer,and Growth of Anabaena sp. (PCC 7119)

Long-term and short-term effects of gramine on cells of Anabaena sp. were studied. Culture death was observed after an initial growth in the presence of 0.5 mM gramine, and lower concentrations decreased both the specific growth rate and the growth yield. Cultures showed a reduction in the chlorophyll content as well as an increase in the level of accessory pigments, which were proportional to the alkaloid concentration. When cultures were excited with green light in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea, the fluorescence spectra of the cells showed a shoulder at 685 nm related to the photosystem II (PSII) antennae emission. This band was reduced when gramine was present during the growth, suggesting that gramine suppresses the energy transfer between the phycobilisomes and PSII. At lethal concentrations for cellular growth, gramine suppressed immediately the photosynthetic oxygen production as well as the electron transport from H2O to p-benzoquinone. The influence of gramine on the PSII photochemical reactions was investigated by flash-induced fluorescence measurements, and the results suggest that the alkaloid could act as an electron donor to the PSII reaction center.     

The primary structure of thioredoxin from the filamentous cyanobacterium Anabaena sp. 7119

Thioredoxin from the cyanobacterium Anabaena 7119 serves as electron donor to ribonucleotide reductase and as a protein disulfide reductase. This small, heat-stable protein was found to have structural and functional similarities to thioredoxins from both bacterial and mammalian sources. We here report the complete primary structure of Anabaena thioredoxin. The structure was determined by analysis of peptides obtained after cleavage with cyanogen bromide, Staphylococcus aureus protease, and trypsin. The protein consists of 106 residues with the following amino acid sequence: Ser-Ala-Ala-Ala-Gln-Val-Thr-Asp- Ser-Thr-Phe-Lys-Gln-Glu-Val-Leu-Asp-Ser-Asp-Val-Pro-Val-leu-Val-Asp-Phe- Trp-Ala-Pro-Trp-Cys-Gly-Pro-Cys-Arg-Met-Val-Ala-Pro-Val-Val-Asp-Glu- Ile-Ala-Gln-Gln-Tyr-Glu-Gly-Lys-Ile-Lys-Val-Val-Lys-Val-Asn-Thr-Asp- Glu-Asn-Pro-Gln-Val-Ala-Ser-Gln-Tyr-Gly-Ile-Arg-Ser-Ile-Pro-Thr-Leu- Met-Ile-Phe-Lys-Gly-Gly-Gln-Lys-Val-Asp-Met-Val-Val-Gly-Ala-Val-Pro- Lys-Thr-Thr-Leu-Ser-Gln-Thr-Leu-Glu-Lys-His-Leu. The sequence of Anabaena thioredoxin shows a definite homology to the protein from Escherichia coli, with 49% residue identities occurring in the proteins when aligned at the active site disulfide.     

Purification and molecular properties of nitrite reductase from Anabaena sp. 7119

Ferredoxin-nitrite reductase (EC 1.7.7.1.) from the cyanobacteria Anabaena sp. 7119 has been purified 763-fold with a specific activity of 21.5 units/nig protein (0.358 μkatals/mg). The enzyme has a molecular mass of 52,000 daltons with a Stokes radius of 3.09 nm and a sedimentation coefficient of 4.07 S. The cellular level of nitrite reductase activity gradually increases in response to the addition of increasing amounts of iron to the culture medium.
When partially purified nitrite reductase preparations are subjected to sucrose-density-gradient centrifugation there is a dose correspondence between nitrite reductase activity and absorbance at 400 nm. This suggests the association of a heme chromophore with the enzyme. Furthermore, the presence of an iron-sulfur center is suggested by a close association of acid-labile sulfide with nitrite reductase activity. Carbon monoxide inhibits nitrite reductase activity. The nature and kinetics of this reaction are comparable to other siroheme-containing nitrite reductases.     

Characterization of cytochrome c 6 from the cyanobacterium Anabaena PCC 7119

 A soluble monoheme c–type cytochrome c ₆ has been isolated from the cyanobacterium Anabaena PCC 7119. It is a basic protein, with a molecular mass of 9.7 kDa, which accepts electrons from Anabaena ferredoxin in the ferredoxin-NADP⁺reductase-dependent NADPH cytochrome c reductase activity assay. The turnover of the reaction has an optimum pH at 7.5. Flavodoxin can also replace ferredoxin in this assay, but with only 20% efficiency. Plastocyanin from Anabaena PCC 7119, as well as the c ₆ cytochromes from the green algae Chlorella fusca and Monoraphidium braunii are also shown to accept electrons from Anabaena ferredoxin. The reduction potential of cytochrome c ₆ at pH 6.7 was determined to be 338 mV and is pH dependent, with pK _a ^ox=8.4±0.1 and pK _a ^red≈9.5. The ferric and ferrous cytochrome forms and their pH equilibria have been studied using visible, EPR and ¹H-NMR spectroscopies. The amino acid sequence and the visible and NMR spectroscopic data indicate that the heme iron has a methionine-histidine axial coordination in the pH range 5–11. However, the EPR data for the ferricytochrome are complex and show that in this pH range five distinct forms are present. Between pH 5 and 9 the spectrum is dominated by two rhombic species, with g–values at 2.94, 2.29, 1.43 and at 2.84, 2.34, 1.56, which interconvert with a pK _a of 8.4. The NMR data also show a main interconversion between two cytochrome forms at this pH, which coincides with that determined from the pH dependence of the reduction potential. Both these forms were associated with a methionine-histidine heme-iron coordination by correlation with the visible and NMR spectral data, although having crystal field parameters atypical for this type of coordination. Anabaena cytochrome c ₆ is one more example of a heme protein for which the widely used crystal field analysis of the EPR data (truth diagram) fails to unequivocally determine the type of heme-iron ligation. Received: 17 May 1996 / Accepted: 13 January 1997     

Anabaena sp. PCC 7119 flavodoxin as electron carrier from photosystem I to ferredoxin-NADP+ reductase. Role of Trp(57) and Tyr(94)

The influence of the amino acid residues sandwiching the flavin ring in flavodoxin (Fld) from the cyanobacterium Anabaena sp. PCC 7119 in complex formation and electron transfer (ET) with its natural partners, photosystem I (PSI) and ferredoxin-NADP(+) reductase (FNR), was examined in mutants of the key residues Trp(57) and Tyr(94). The mutants' ability to form complexes with either FNR or PSI is similar to that of wild-type Fld. However, some of the mutants exhibit altered kinetic properties in their ET processes that can be explained in terms of altered flavin accessibility and/or thermodynamic parameters. The most noticeable alteration is produced upon replacement of Tyr(94) by alanine. In this mutant, the processes that involve the transfer of one electron from either PSI or FNR are clearly accelerated, which might be attributable to a larger accessibility of the flavin to the reductant. However, when the opposite ET flow is analyzed with FNR, the reduced Y94A mutant transfers electrons to FNR slightly more slowly than wild type. This can be explained thermodynamically from a decrease in driving force due to the significant shift of 137 mV in the reduction potential value for the semiquinone/hydroquinone couple (E(1)) of Y94A, relative to wild type (Lostao, A., Gómez-Moreno, C., Mayhew, S. G., and Sancho, J. (1997) Biochemistry 36, 14334-14344). The behavior of the rest of the mutants can be explained in the same way. Overall, our data indicate that Trp(57) and Tyr(94) do not play any active role in flavodoxin redox reactions providing a path for the electrons but are rather involved in setting an appropriate structural and electronic environment that modulates in vivo ET from PSI to FNR while providing a tight FMN binding.     

CW-EPR and ENDOR study of cytochrome c6 from Anabaena PCC 7119

The detailed analysis of the continuous-wave electron paramagnetic resonance and electron nuclear double resonance measurements on cytochrome c(6) from Anabaena PCC7119 reveals several electronic and structural properties of this hemeprotein. The oxidized protein shows two forms that differ in the arrangement of the residues that act as heme axial ligands. Information about the orientation of these residues is obtained for one of the forms, which turns out to differ from that found in the reduced protein from x-ray experiments. The biological significance of these results is discussed.     

Functional characterization of the dimerization domain of the ferric uptake regulator (Fur) of Pseudomonas aeruginosa

The functional properties of the recombinant C-terminal dimerization domain of the Pseudomonas aeruginosa Fur (ferric uptake regulator) protein expressed in and purified from Escherichia coli have been evaluated. Sedimentation velocity measurements demonstrate that this domain is dimeric, and the UV CD spectrum is consistent with a secondary structure similar to that observed for the corresponding region of the crystallographically characterized wild-type protein. The thermal stability of the domain as determined by CD spectroscopy decreases significantly as pH is increased and increases significantly as metal ions are added. Potentiometric titrations (pH 6.5) establish that the domain possesses a high-affinity and a low-affinity binding site for metal ions. The high-affinity (sensory) binding site demonstrates association constants (K(A)) of 10(+/-7)x10(6), 5.7(+/-3)x10(6), 2.0(+/-2)x10(6) and 2.0(+/-3)x10(4) M(-1) for Ni2+, Zn2+, Co2+ and Mn2+ respectively, while the low-affinity (structural) site exhibits association constants of 1.3(+/-2)x10(6), 3.2(+/-2)x10(4), 1.76(+/-1)x10(5) and 1.5(+/-2)x10(3) M(-1) respectively for the same metal ions (pH 6.5, 300 mM NaCl, 25 degrees C). The stability of metal ion binding to the sensory site follows the Irving-Williams order, while metal ion binding to the partial sensory site present in the domain does not. Fluorescence experiments indicate that the quenching resulting from binding of Co2+ is reversed by subsequent titration with Zn2+. We conclude that the domain is a reasonable model for many properties of the full-length protein and is amenable to some analyses that the limited solubility of the full-length protein prevents.     

A single arginyl residue in plastocyanin and in cytochrome c(6) from the cyanobacterium Anabaena sp. PCC 7119 is required for efficient reduction of photosystem I

Positively charged plastocyanin from Anabaena sp. PCC 7119 was investigated by site-directed mutagenesis. The reactivity of its mutants toward photosystem I was analyzed by laser flash spectroscopy. Replacement of arginine at position 88, which is adjacent to the copper ligand His-87, by glutamine and, in particular, by glutamate makes plastocyanin reduce its availability for transferring electrons to photosystem I. Such a residue in the copper protein thus appears to be isofunctional with Arg-64 (which is close to the heme group) in cytochrome c(6) from Anabaena (Molina-Heredia, F. P., Diaz-Quintana, A., Hervás, M., Navarro, J. A., and De la Rosa, M. A. (1999) J. Biol. Chem. 274, 33565-33570) and Synechocystis (De la Cerda, B., Diaz-Quintana, A., Navarro, J. A. , Hervás, M., and De la Rosa, M. A. (1999) J. Biol. Chem. 274, 13292-13297). Other mutations concern specific residues of plastocyanin either at its positively charged east face (D49K, H57A, H57E, K58A, K58E, Y83A, and Y83F) or at its north hydrophobic pole (L12A, K33A, and K33E). Mutations altering the surface electrostatic potential distribution allow the copper protein to modulate its kinetic efficiency: the more positively charged the interaction site, the higher the rate constant. Whereas replacement of Tyr-83 by either alanine or phenylalanine has no effect on the kinetics of photosystem I reduction, Leu-12 and Lys-33 are essential for the reactivity of plastocyanin.     

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

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