The solution structure of the recombinant hemoglobin from the cyanobacterium Synechocystis sp. PCC 6803 in its hemichrome state

The product of the cyanobacterium Synechocystis sp. PCC 6803 gene slr2097 is a 123 amino acid polypeptide chain belonging to the truncated hemoglobin family. Recombinant, ferric heme-reconstituted Synechocystis sp. PCC 6803 hemoglobin displays bis-histidine coordination of the iron ion. In addition, this protein is capable of covalently attaching a reactive histidine to the heme 2-vinyl group. The structure of the protein in the low-spin ferric state with intact vinyl substituents was solved by NMR methods. It was found that the structure differs from that of known truncated hemoglobins primarily in the orientation of the E helix, which carries His46 (E10) as the distal ligand to the iron; the length and orientation of the F helix, which carries His70 (F8) as the proximal ligand to the iron; and the H-helix, which carries His117 (H16), the reactive histidine. Regions of enhanced flexibility include the short A helix, the loop connecting the E and F helices, and the last seven residues at the carboxy end. The structural data allowed for the rationalization of physical properties of the cyanobacterial protein, such as fast on-rate for small ligand binding, unstable apoprotein fold, and cross-linking ability. Comparison to the truncated hemoglobin from the green alga Chlamydomonas eugametos also suggested how the endogenous hexacoordination affected the structure.     

Binding of ferric heme by the recombinant globin from the cyanobacterium Synechocystis sp. PCC 6803

The product of the cyanobacterium Synechocystis sp. PCC 6803 gene slr2097 is a 123 amino acid polypeptide chain belonging to the truncated hemoglobin family. Recombinant, ferric heme-reconstituted Synechocystis sp. PCC 6803 hemoglobin is a low-spin complex whose endogenous hexacoordination gives rise to optical and NMR characteristics reminiscent of cytochrome b(5) [Scott, N. L., and Lecomte, J. T. J. (2000) Protein Sci. 9, 587-597]. In this work, the sequential assignments using (15)N-(13)C-labeled protein, (1)H nuclear Overhauser effects, and longitudinal relaxation data identified His70 as the proximal histidine and His46 as the sixth ligand to the iron ion. It was also found that one of two possible heme orientations within the protein matrix is highly preferred (>90%) and that this orientation is the same as in vertebrate myoglobins. The rate constant for the 180 degrees rotation of the heme within a protein cage to produce the favored isomer was 0.5 h(-1) at 25 degrees C, approximately 35 times faster than in sperm whale myoglobin. Variable temperature studies revealed an activation energy of 132 +/- 4 kJ mol(-1), similar to the value in metaquomyoglobin at the same pH. The rate constant for heme loss from the major isomer was estimated to be 0.01 h(-1) by optical spectroscopy, close to the value for myoglobin and decades slower than in the related Nostoc commune cyanoglobin. The slow heme loss was attributed in part to the additional coordination bond to His46, whereas the relatively fast rate of heme reorientation suggested that this bond was weaker than the proximal His70-Fe bond. The standard reduction potential of the hexacoordinated protein was measured with and without poly-L-lysine as a mediator and found to be approximately -150 mV vs SHE, indicating a stabilization of the ferric state compared to most hemoglobins and b(5) cytochromes.     

Cyanide binding to hexacoordinate cyanobacterial hemoglobins: hydrogen-bonding network and heme pocket rearrangement in ferric H117A Synechocystis hemoglobin

The truncated hemoglobin (Hb) from the cyanobacterium Synechocystis sp. PCC 6803 is a bis-histidyl hexacoordinate complex in the absence of exogenous ligands. This protein can form a covalent cross-link between His117 in the H-helix and the heme 2-vinyl group. Cross-linking, the physiological importance of which has not been established, is avoided with the His117Ala substitution. In the present work, H117A Hb was used to explore exogenous ligand binding to the heme group. NMR and thermal denaturation data showed that the replacement was of little consequence to the structural and thermodynamic properties of ferric Synechocystis Hb. It did, however, decelerate the association of cyanide ions with the heme iron. Full complexation required hours, instead of minutes, of incubation at optical and NMR concentrations. At neutral pH and in the presence of excess cyanide, binding occurred with a first-order dependence on cyanide concentration, eliminating distal histidine decoordination as the rate-limiting step. The cyanide complex of the H117A variant was characterized for the conformational changes occurring as the histidine on the distal side, His46 (E10), was displaced. Extensive rearrangement allowed Tyr22 (B10) to insert in the heme pocket and Gln43 (E7) and Gln47 (E11) to come in contact with it. H-bond formation to the bound cyanide was identified in solution with the use of (1)H(2)O/(2)H(2)O mixtures. Cyanide binding also resulted in a change in the ratio of heme orientational isomers, in a likely manifestation of heme environment reshaping. Similar observations were made with the related Synechococcus sp. PCC 7002 H117A Hb, except that cyanide binding was rapid in this protein. In both cases, the (15)N chemical shift of bound cyanide was reminiscent of that in peroxidases and the orientation of the proximal histidine was as in other truncated Hbs. The ensemble of the data provided insight into the structural cooperativity of the heme pocket scaffold and pointed to the reactive 117 site of Synechocystis Hb as a potential determinant of biophysical and, perhaps, functional properties.     

Cloning, expression, purification, and preliminary characterization of a putative hemoglobin from the cyanobacterium Synechocystis sp. PCC 6803

The genome of the unicellular cyanobacterium Synechocystis sp. PCC 6803 contains a gene (slr2097, glbN) encoding a 123 amino-acid product with sequence similarity to globins. Related proteins from cyanobacteria, ciliates, and green algae bind oxygen and have a pronounced tendency to coordinate the heme iron with two protein ligands. To study the structural and functional properties of Synechocystis sp. PCC 6803 hemoglobin, slr2097 was cloned and overexpressed in Escherichia coli. Purification of the hemoglobin was performed after addition of hemin to the clarified cell lysate. Recombinant, heme-reconstituted ferric Synechocystis sp. PCC 6803 hemoglobin was found to be a stable helical protein, soluble to concentrations higher than 500 microM. At neutral pH, it yielded an electronic absorption spectrum typical of a low-spin ferric species, with maxima at 410 and 546 nm. The proton NMR spectrum revealed sharp lines spread over a chemical shift window narrower than 40 ppm, in support of low-spin hexacoordination of the heme iron. Nuclear Overhauser effects demonstrated that the heme is inserted in the protein matrix to produce one major equilibrium form. Addition of dithionite resulted in an absorption spectrum with maxima at 426, 528, and 560 nm. This reduced form appeared capable of carbon monoxide binding. Optical data also suggested that cyanide ions could bind to the heme in the ferric state. The spectral properties of the putative Synechocystis sp. PCC 6803 hemoglobin confirmed that it can be used for further studies of an ancient hemoprotein structure.     

Structural and dynamic properties of Synechocystis sp. PCC 6803 Hb revealed by reconstitution with Zn-protoporphyrin IX

In its resting state, the truncated globin of the cyanobacterium Synechocystis sp. PCC 6803 exhibits hexacoordination of the heme iron, with His46 (E10) and His70 (F8, proximal) serving as axial ligands. Diatomic ligands displace the distal His46 (E10) from the ferric and ferrous iron and promote considerable structural changes in the B helix, E helix, and EF regions. Here, Zn(II)-substituted hemoglobin was used to explore the role of distal ligands in stabilizing the heme pocket structure. NMR data showed that the Zn ion was coordinated by the four pyrrole nitrogens and by His70 (F8) only. The proximal side of the Zn-porphyrin adopted a geometry recognizable as that of the wild-type protein. Decoordination of His46 (E10) to form the pentacoordinate Zn resulted in an incomplete transition to the conformation observed in the ferric, cyanide-bound protein. The NMR data also demonstrated that the H helix underwent complex dynamic processes near His117, a residue readily reacting with the wild-type heme 2-vinyl group in a post-translational modification.     

Proximal influences in two-on-two globins: effect of the Ala69Ser replacement on Synechocystis sp. PCC 6803 hemoglobin

The cyanobacterium Synechocystis sp. PCC 6803 (S6803) expresses a two-on-two globin in which His46 (distal side) and His70 (proximal) function as heme iron axial ligands. His46 can be displaced by O2, CO, and CN-, among others, whereas His70 is not labile under native conditions. The residue preceding the proximal histidine has been implicated in controlling globin axial ligand reactivity; the details of the mechanism, however, are not well understood, and little information exists for bis-histidyl hexacoordinate proteins. In many vertebrate hemoglobins and in the Synechocystis protein, the position is occupied by an alanine, whereas, in myoglobins, it is a serine involved in an intricate hydrogen-bond network. We examined the role of Ala69 in S6803 hemoglobin through the effects of an Ala --> Ser replacement. The substitution resulted in minor structural perturbations, but the response of the holoprotein to temperature-, urea-, and acid-induced denaturation was measurably affected. Enhanced three-state behavior was manifested in the decoupling of heme binding and secondary-structure formation. Urea-gradient gel experiments revealed that the stability of the apoprotein was unchanged by the replacement and that a slight alteration of the folding kinetics occurred in the holoproteins. Cyanide-binding experiments were performed to assess trans effects. The apparent rate constant for association decreased 2-fold upon Ala69Ser replacement. This deceleration was attributed to a change in the lifetime of a state containing a decoordinated His46. The results demonstrated that, as in vertebrate globins and leghemoglobin, proximal influences operate to determine fundamental dynamic and thermodynamic properties of the protein.     

Subcellular localization of the BtpA protein in the cyanobacterium Synechocystis sp. PCC 6803.

Photosystem I is a large pigment-protein complex embedded in the thylakoid membranes of chloroplasts and cyanobacteria. In the cyanobacterium Synechocystis sp. PCC 6803, the btpA gene encodes a 30-kDa polypeptide. Mutations in this gene significantly affect accumulation of the reaction center proteins of photosystem I in Synechocystis 6803 [Bartsevich, V. V. & Pakrasi, H. B. (1997) J. Biol. Chem. 272, 6372-6378]. We describe here the intracellular localization of the BtpA protein. Immunolocalization in Synechocystis 6803 cells demonstrated that the BtpA protein is tightly associated with the thylakoid membranes. Phase fractionation in the detergent Triton X-114 indicated that BtpA is a peripheral membrane protein. To determine which surface of the thylakoid membrane BtpA is exposed to, we used a two-phase polymer partitioning technique to develop a novel method to isolate inside-out and right-side-out thylakoid vesicles from Synechocystis 6803. Treatments of such vesicles with different salts and protease showed that the BtpA protein is an extrinsic membrane protein which is exposed to the cytoplasmic face of the thylakoid membrane.     

Effect of increased PHA synthase activity on polyhydroxyalkanoates biosynthesis in Synechocystis sp. PCC6803

Polyhydroxyalkanoate (PHA) synthase activity in Synechocystis sp. PCC6803 was increased two-fold by introducing the PHA biosynthetic genes of Ralstonia eutropha. The resulting recombinant Synechocystis sp. PCC6803 strain was subjected to conditions that favor PHA accumulation and the effects of various carbon sources were studied. In addition, the fine structure of both wild-type and recombinant Synechocystis sp. PCC6803 was examined using freeze-fracture electron microscopy technique. The PHA granules in the recombinant Synechocystis sp. PCC6803 were localised near the thylakoid membranes. Maximum amount of PHA accumulation was obtained in the presence of acetate, where the number of granules in the recombinant cells ranged from 4 to 6 and their sizes were in the range of 70-240 nm. In comparison to wild-type Synechocystis sp. PCC6803, recombinant cells with increased PHA synthase activity showed only a marginal increase in PHA content suggesting that PHA synthase is not the rate limiting enzyme of PHA biosynthesis in Synechocystis sp. PCC6803.     

Targeted random mutagenesis to identify functionally important residues in the D2 protein of photosystem II in Synechocystis sp. strain PCC 6803

To identify important residues in the D2 protein of photosystem II (PSII) in the cyanobacterium Synechocystis sp. strain PCC 6803, we randomly mutagenized a region of psbDI (coding for a 96-residue-long C-terminal part of D2) with sodium bisulfite. Mutagenized plasmids were introduced into a Synechocystis sp. strain PCC 6803 mutant that lacks both psbD genes, and mutants with impaired PSII function were selected. Nine D2 residues were identified that are important for PSII stability and/or function, as their mutation led to impairment of photoautotrophic growth. Five of these residues are likely to be involved in the formation of the Q(A)-binding niche; these are Ala249, Ser254, Gly258, Ala260, and His268. Three others (Gly278, Ser283, and Gly288) are in transmembrane alpha-helix E, and their alteration leads to destabilization of PSII but not to major functional alterations of the remaining centers, indicating that they are unlikely to interact directly with cofactors. In the C-terminal lumenal tail of D2, only one residue (Arg294) was identified as functionally important for PSII. However, from the number of mutants generated it is likely that most or all of the 70 residues that are susceptible to bisulfite mutagenesis have been altered at least once. The fact that mutations in most of these residues have not been picked up by our screening method suggests that these mutations led to a normal photoautotrophic phenotype. A novel method of intragenic complementation in Synechocystis sp. strain PCC 6803 was developed to facilitate genetic analysis of psbDI mutants containing several amino acid changes in the targeted domain. Recombination between genome copies in the same cell appears to be much more prevalent in Synechocystis sp. strain PCC 6803 than was generally assumed.     

Purification and characterization of class-I and class-II fructose-1,6-bisphosphate aldolases from the cyanobacterium Synechocystis sp. PCC 6803

The whole genome sequence database for Synechocystis sp. PCC 6803 has revealed the presence of genes encoding class-I (CI) and class-II (CII) fructose-1,6-bisphosphate aldolases (FBAs) in this organism. Two types of FBA from Synechocystis sp. PCC 6803 were separated by chromatography on phenyl-Sepharose. The activity of the enzyme in the major peak was inhibited by the presence of 25 mM EDTA; however, the activity in the minor peak was not. Therefore, the FBA in the former fractions was designated as CII-FBA, and in the latter designated as CI-FBA. CI-FBA was functionally redundant in Synechocystis sp. PCC 6803, while no disruptant for the gene encoding CII-FBA was obtained under photoautotrophic conditions. The kinetic parameters of CI- and CII-FBAs purified from Synechocystis sp. PCC 6803 in the cleavage reaction of FBP were generally similar, except in their reactivity for SBP. The SBP/FBP activity ratio of the CII-FBA was two times higher than that of the CI-FBA.     

Chemical reactivity of <Emphasis Type="Italic">Synechococcus</Emphasis> sp. PCC 7002 and <Emphasis Type="Italic">Synechocystis</Emphasis> sp. PCC 6803 hemoglobins: covalent heme attachment and bishistidine coordination

In the absence of an exogenous ligand, the hemoglobins from the cyanobacteria Synechocystis sp. PCC 6803 and Synechococcus sp. PCC 7002 coordinate the heme group with two axial histidines (His46 and His70). These globins also form a covalent linkage between the heme 2-vinyl substituent and His117. The in vitro mechanism of heme attachment to His117 was examined with a combination of site-directed mutagenesis, NMR spectroscopy, and optical spectroscopy. The results supported an electrophilic addition with vinyl protonation being the rate-determining step. Replacement of His117 with a cysteine demonstrated that the reaction could occur with an alternative nucleophile. His46 (distal histidine) was implicated in the specificity of the reaction for the 2-vinyl group as well as protection of the protein from oxidative damage caused by exposure to exogenous H₂O₂.     

Ssr2998 of Synechocystis sp. PCC 6803 is involved in regulation of cyanobacterial electron transport and associated with the cytochrome b6f complex

To analyze the function of a protein encoded by the open reading frame ssr2998 in Synechocystis sp. PCC 6803, the corresponding gene was disrupted, and the generated mutant strain was analyzed. Loss of the 7.2-kDa protein severely reduced the growth of Synechocystis, especially under high light conditions, and appeared to impair the function of the cytochrome b6 f complex. This resulted in slower electron donation to cytochrome f and photosystem 1 and, concomitantly, over-reduction of the plastoquinone pool, which in turn had an impact on the photosystem 1 to photosystem 2 stoichiometry and state transition. Furthermore, a 7.2-kDa protein, encoded by the open reading frame ssr2998, was co-isolated with the cytochrome b6 f complex from the cyanobacterium Synechocystis sp. PCC 6803. ssr2998 seems to be structurally and functionally associated with the cytochrome b6 f complex from Synechocystis, and the protein could be involved in regulation of electron transfer processes in Synechocystis sp. PCC 6803.     

Substrate specificities and availability of fucosyltransferase and beta-carotene hydroxylase for myxol 2'-fucoside synthesis in Anabaena sp. strain PCC 7120 compared with Synechocystis sp. strain PCC 6803

To elucidate the biosynthetic pathways of carotenoids, especially myxol 2'-glycosides, in cyanobacteria, Anabaena sp. strain PCC 7120 (also known as Nostoc sp. strain PCC 7120) and Synechocystis sp. strain PCC 6803 deletion mutants lacking selected proposed carotenoid biosynthesis enzymes and GDP-fucose synthase (WcaG), which is required for myxol 2'-fucoside production, were analyzed. The carotenoids in these mutants were identified using high-performance liquid chromatography, field desorption mass spectrometry, and (1)H nuclear magnetic resonance. The wcaG (all4826) deletion mutant of Anabaena sp. strain PCC 7120 produced myxol 2'-rhamnoside and 4-ketomyxol 2'-rhamnoside as polar carotenoids instead of the myxol 2'-fucoside and 4-ketomyxol 2'-fucoside produced by the wild type. Deletion of the corresponding gene in Synechocystis sp. strain PCC 6803 (sll1213; 79% amino acid sequence identity with the Anabaena sp. strain PCC 7120 gene product) produced free myxol instead of the myxol 2'-dimethyl-fucoside produced by the wild type. Free myxol might correspond to the unknown component observed previously in the same mutant (H. E. Mohamed, A. M. L. van de Meene, R. W. Roberson, and W. F. J. Vermaas, J. Bacteriol. 187:6883-6892, 2005). These results indicate that in Anabaena sp. strain PCC 7120, but not in Synechocystis sp. strain PCC 6803, rhamnose can be substituted for fucose in myxol glycoside. The beta-carotene hydroxylase orthologue (CrtR, Alr4009) of Anabaena sp. strain PCC 7120 catalyzed the transformation of deoxymyxol and deoxymyxol 2'-fucoside to myxol and myxol 2'-fucoside, respectively, but not the beta-carotene-to-zeaxanthin reaction, whereas CrtR from Synechocystis sp. strain PCC 6803 catalyzed both reactions. Thus, the substrate specificities or substrate availabilities of both fucosyltransferase and CrtR were different in these species. The biosynthetic pathways of carotenoids in Anabaena sp. strain PCC 7120 are discussed.     

From genome to enzyme: analysis of key glycolytic and oxidative pentose-phosphate pathway enzymes in the cyanobacterium Synechocystis sp. PCC 6803

Activities of glucokinase, glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, phosphoglucose isomerase, phosphofructokinase (PFK), enolase, pyruvate kinase (PK) and phosphoenolpyruvate (PEP) carboxylase were determined in extracts of photoautotrophic, mixotrophic, and heterotrophic cultures of Synechocystis sp. PCC 6803. Annotated genomes of Synechocystis sp. PCC 6803 and Anabaena sp. PCC 7120 were analyzed for the respective predicted physical properties of each enzyme investigated here. Enzymatic activity was largely unaffected by nutritional mode, with the exception of glucokinase and PK whose activities were significantly elevated in heterotrophic cultures of Synechocystis sp. PCC 6803. PFK activity was insensitive to bacterial PFK-A (allosteric) effectors such as PEP, implying that Synechocystis PFK should be classified as a PFK-B (non-allosteric). Immunoblot and kinetic studies indicated that irrespective of nutritional mode, the Synechocystis PK corresponds to a PK-A (AMP activated) rather than PK-F (fructose-1,6-bisphosphate activated).     

phrA, the major photoreactivating factor in the cyanobacterium Synechocystis sp. strain PCC 6803 codes for a cyclobutane-pyrimidine-dimer-specific DNA photolyase

A new broad-host-range plasmid, pSL1211, was constructed for the over-expression of genes in Synechocystis sp. strain PCC 6803. The plasmid was derived from RSF1010 and an Escherichia coli over-expression plasmid, pTrcHisC. Over-expressed protein is made with a removable N-terminal histidine tag. The plasmid was used to over-express the phrA gene and purify the gene product from Synechocystis sp. strain PCC 6803. PhrA is the major ultraviolet-light-resistant factor in the cyanobacterium. The purified PhrA protein exhibited an optical absorption spectrum similar to that of the cyclobutane pyrimidine dimer (CPD) DNA photolyase from Synechocuccus sp. strain PCC 6301 (Anacystis nidulans). Mass spectrometry analysis of PhrA indicated that the protein contains 8-hydroxy-5-deazariboflavin and flavin adenine dinucleotide (FADH2) as cofactors. PhrA repairs only cyclobutane pyrimidine dimer but not pyrimidine (6-4) pyrimidinone photoproducts. On the basis of these results, the PhrA protein is classified as a class I, HDF-type, CPD DNA photolyase.