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J. Biol. Chem., Vol. 277, Issue 46, 43615-43622, November 15, 2002

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Biochemical Characterization of a Membrane-bound Manganese-containing Superoxide Dismutase from the Cyanobacterium Anabaena PCC 7120^*

Günther Regelsberger, Werner Atzenhofer^§, Florian Rüker^¶, Günter A. Peschek, Christa Jakopitsch, Martina Paumann, Paul Georg Furtmüller, and Christian Obinger^**

From the  Institute of Chemistry, University of Agricultural Sciences, Muthgasse 18, A-1190 Vienna, Austria, ^§ Max-Planck-Institut für Biochemie/Abteilung Strukturforschung, Am Klopferspitz 18a, D-82152 Martinsried/Planegg-München, Germany, the ^¶ Institute of Applied Microbiology, University of Agricultural Sciences, Muthgasse 18, A-1190 Vienna, Austria, and the  Institute of Physical Chemistry, Molecular Bioenergetics Group, University of Vienna, Althanstrasse 14, A-1090 Vienna, Austria

Received for publication, July 30, 2002, and in revised form, August 29, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The filamentous cyanobacterium Anabaena PCC 7120 (now renamed Nostoc PCC 7120) possesses two genes for superoxide dismutase (SOD). One is an iron-containing (FeSOD) whereas the other is a manganese-containing superoxide dismutase (MnSOD). Localization experiments and analysis of the sequence showed that the FeSOD is cytosolic, whereas the MnSOD is a membrane-bound homodimeric protein containing one transmembrane helix, a spacer region, and a soluble catalytic domain. It is localized in both cytoplasmic and thylakoid membranes at the same extent with the catalytic domains positioned either in the periplasm or the thylakoid lumen. A phylogenetic analysis revealed that generally the highly homologous MnSODs of filamentous cyanobacteria are unique in being membrane-bound. Two recombinant variants of Anabaena MnSOD lacking either the hydrophobic region (MnSOD(28)) or the hydrophobic and the linker region (MnSOD(60)) are shown to exhibit the characteristic manganese peak at 480 nm, an almost 100% occupancy of manganese per subunit, a specific activity using the ferricytochrome assay of (660 ± 90) unit mg¹ protein and a dissociation constant for the inhibitor azide of (0.84 ± 0.05) mM. Using stopped-flow spectroscopy it is shown that the decay of superoxide in the presence of various (MnSOD(28)) or (MnSOD(60)) concentrations is first-order in enzyme concentration allowing the calculation of catalytic rate constants which increase with decreasing pH: 8 × 10⁶ M¹ s¹ (pH 10) and 6 × 10⁷ M¹ s¹ (pH 7). The physiological relevance of these findings is discussed with respect to the bioenergetic peculiarities of cyanobacteria.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Cyanobacteria, which were also called blue-green algae, are primordial organisms evolving more than three billion years ago. These organisms form one of the main eubacterial phyla and one of the largest groups of Gram-negative bacteria. Through endosymbiotic processes ancient cyanobacteria were the progenitors of plant plastids. Today nearly 2000 cyanobacterial species are known with a variety of physiological, morphological, and developmental properties and colonizing a wide range of habitats. They are capable of performing oxygenic photosynthesis and are clearly distinguished from the other photosynthetic bacteria such as purple and green bacteria because cyanobacteria use water as an electron donor thereby releasing molecular oxygen into the environment. Because of the high intracellular oxygen concentration produced during photosynthesis potentially damaging reactive oxygen species can be formed. Two pathways can lead to this toxic molecule. One pathway produces singlet dioxygen (¹O₂) through interaction of molecular oxygen with excited triplet-state chlorophyll. This singlet dioxygen is especially harmful to polyunsaturated fatty acids leading to lipid peroxidation and membrane damage. Carotenoids are able to quench the formation of triplet-state chlorophyll (1). The second pathway of molecular oxygen activation results in the formation of the superoxide radical (O) by interaction with electron acceptors of photosystem I or reduced ferredoxin (2). The superoxide radical is not very reactive in aqueous solution, but it is implicated in lipid peroxidation, protein inactivation (e.g. destruction of iron-sulfur proteins), and formation of the highly reactive hydroxyl radical (^·OH) by reaction with hydrogen peroxide (H₂O₂) (2). To prevent photooxidative damage both hydrogen peroxide and superoxide radical must be scavenged effectively by phototrophic organisms. In solution superoxide can react with a second molecule of superoxide thereby dismutating to hydrogen peroxide and dioxygen. This reaction can also be accomplished by an enzymatic process via proteins called superoxide dismutases (SODs)¹ that lower the level of superoxide. Today four types of superoxide dismutase are known, each containing a different metal ion at the active site: iron (FeSOD), manganese (MnSOD), copper/zinc (CuZnSOD), and nickel (NiSOD). Whereas FeSOD and MnSOD are very similar in sequence and structure, the others show no correspondence. There are several papers that describe superoxide dismutase activities and localization in cyanobacterial cells (3-10). DNA sequences of FeSODs and MnSODs in cyanobacteria are reported (10-13). These reports as well as genome analysis (www.kazusa.or.jp/cyano/) show that cyanobacteria possess either one or more SOD genes. The unicellular cyanobacterium Synechocystis PCC 6803 contains a single FeSOD gene, Plectonema boryanum contains one FeSOD gene as well as three additional MnSOD genes (12) and Nostoc punctiforme contains one FeSOD gene and two MnSOD genes. The few reports about the physiological role of cyanobacterial SODs suggest that these enzymes protect cellular components during oxidative stress or from the effects of chilling. In Nostoc commune a significant role for an extracellular FeSOD in response to desiccation has been proposed (10).

We chose the filamentous, nitrogen-fixing cyanobacterium Anabaena PCC 7120 (now renamed Nostoc PCC 7120) as a model to characterize the equipment for superoxide detoxification. Superoxide dismutase assays have shown that both membrane preparations and cytosolic extracts contain SOD activity. Screening of the genome of Anabaena PCC 7120 revealed that there are two genes for SODs, one has a high homology to iron-containing (sodB gene) and the other to manganese-containing superoxide dismutase (sodA gene). In this paper we report localization of the MnSOD in both cytoplasmic and thylakoid membranes. It is shown that Anabaena MnSOD is unique in being a type 2 membrane-bound protein. Its heterologous overexpression is reported as well as its comprehensive biochemical and kinetic characterization including the determination of actual bimolecular rate constants at pH 7-10.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Materials-- Materials were acquired from the following sources: SigmaMarker (wide molecular weight range), cytochrome c (from horse heart), xanthine, xanthine oxidase (from buttermilk grade I), 18-crown-6-ether, 3-Å molecular sieve, and potassium superoxide from Sigma; the GFX PCR DNA and Gel Band Purification Kit, Gel Filtration LMW calibration kit, Chelating-Sepharose Fast Flow, Superdex 200 prep grade, and PD-10 desalting columns from Amersham Biosciences; the Centriprep-30 concentrators from Amicon; alkaline phosphatase from Roche Molecular Biochemicals; T4 DNA ligase, NdeI, BamHI, and BglII restriction enzymes from MBI Fermentas, and DyNAzyme EXT DNA polymerase from Finnzyme. All other reagents were of highest grade available.

Methods--

Growth Conditions and Membrane Separation-- Axenic cultures of Anabaena PCC 7120 were grown photoautotrophically in BG-11 medium (14) at 35 °C for 1 week under illumination and bubbled with filtered air containing 1% (v/v) of CO₂. Cells were harvested by centrifugation (4000 × g, 10 min, 4 °C), washed twice with Hepes-EDTA buffer (10 mM Hepes/NaOH, pH 7.4, 5 mM NaCl, 2 mM Na₂EDTA) and resuspended in the same buffer to a cell density of 80 µl/ml packed cells. The suspension was made up to 20% (w/w) sucrose and 0.5% lysozyme, incubated at 37 °C for 2 h and centrifuged (3000 × g, 10 min, 4 °C). Thereafter, the pellet was resuspended in Hepes-EDTA buffer containing 0.0075% DNase I, incubated on ice for 30 min, passed twice through a precooled French pressure cell (Aminco) at 40 and 70 megapascals, respectively, and centrifuged (5000 × g, 10 min, 4 °C). The supernatant was used to separate membranes and cytosolic components by ultracentrifugation (250,000 × g, 50 min, 4 °C) or adjusted to 42% (w/w) with solid sucrose for density gradient centrifugation. This solution was overlaid with 35, 30, and 10% (w/w) sucrose in Hepes/NaOH buffer and centrifuged (Beckman SW-27 rotor, 131,500 × g, 16 h, 4 °C). All the fractions obtained by ultracentrifugation were recentrifuged severalfold to reduce cross-contamination, and all buffers contained 1 mM phenylmethylsulfonyl fluoride, 5 µM leupeptin, and 5 µM pepstatin (15).

Overexpression of MnSOD-- DNA and protein sequence of the manganese-containing superoxide dismutase was obtained from the CyanoBase, the genome data base for Anabaena PCC 7120, at www.kazusa.or.jp/cyano/Anabaena/. The following synthetic oligonucleotide primers were constructed and purchased from geneXpress (Maria Wörth, Austria): primer 1a (5'-GGG AAT TCC ATA TGA AAT CAT CTC TGT GGC AA-3'), primer 1b (5'-GGG AAT TCC ATA TGG CAG CAA ATT CCC TGC CGA CTA AC-3'), primer 1c (5'-GGG AAT TCC ATA TGG GTA CAA ATC CAG CCG AGT TAC CA-3'; all of these three primers contain an ATG start codon and an NdeI restriction site), and primer 2 (5'-GGA AGA TCT CTA ATG GTG GTG ATG GTG GTG AGA ATT ACT TTG CCG TGA AGC-3'; 51 bases with a TAG termination codon, a sequence coding for a hexa-histidine tag and a BglII restriction site). One of the first primers and primer 2, cell suspension from Anabaena as the template, and the DyNAzyme EXT DNA polymerase were used for PCR under the following conditions: 94 °C for 2 min (hot start); 30 cycles at 92 °C for 1 min, 49 °C for 1 min, and 72 °C for 2 min; and finally at 72 °C for 10 min. The PCR product was fractionated on a 1% agarose gel, and the appropriate DNA was cut out and purified using the GFX PCR DNA and Gel Band Purification Kit. This DNA fragment was digested with the restriction enzymes NdeI and BglII and cloned into the NdeI- and BamHI-digested, alkaline phosphatase-treated expression vector pET-3a (16). The insert was sequenced by the dideoxy chain termination method (17). Competent Escherichia coli BL21(DE3)pLysS was transformed with the expression vector by electroporation (Gene Pulser, Bio-Rad), positive clones carrying the recombinant plasmid were selected and grown overnight on an orbital shaker (180 rpm) at 37 °C in LB medium containing 100 µg/ml ampicillin and 25 µg/ml chloramphenicol. After dilution of the cell suspension with M9ZB medium containing the same antibiotics and 5 mM MnCl₂, expression of superoxide dismutase was induced by the addition of 1 mM isopropyl -D-thiogalactopyranoside. After 4 h of incubation at 37 °C, cells were harvested by centrifugation (5000 × g, 5 min, 4 °C), frozen, and stored at -80 °C until used. Thawed cell paste was resuspended in lysis buffer (50 mM Tris/HCl, pH 8.0, containing 2 mM EDTA and 0.1% Triton X-100), phenylmethylsulfonyl fluoride (1 mM), leupeptin (5 µM), and pepstatin (5 µM) were added, and the cells were broken by sonication with short bursts. The suspension was centrifuged (21,000 × g, 20 min, 4 °C), and from the supernatant the superoxide dismutase was purified.

Protein Purification-- The supernatant was adjusted to 1 M NaCl and 20 mM imidazole and loaded on a chelating-Sepharose Fast Flow column (1.6 × 10 cm) charged with 30 µmol Zn²⁺/ml gel and equilibrated with 67 mM phosphate buffer, pH 7.0, containing 1 M NaCl and 20 mM imidazole at 4 °C. The column was washed with 200 ml of the equilibration buffer, and bound proteins were eluted with 100 ml of a gradient of 20-500 mM imidazole in 67 mM phosphate buffer, pH 7.0, containing 1 M NaCl. The eluted proteins were concentrated, and the buffer was exchanged with Centriprep concentrators. Proteins were loaded onto a Superdex 200 column equilibrated with 67 mM phosphate buffer, pH 7.0, containing 150 mM KCl at room temperature. Active fractions were concentrated using Centriprep concentrators. For molecular mass determination the Superdex 200 column was calibrated with the Gel Filtration LMW calibration kit in the range of 13.7-67 kDa.

Polyacrylamide Gel Electrophoresis-- SDS-PAGE was carried out on 15% slab gels as described by Laemmli (18) using SigmaMarkers and the Bio-Rad Mini-Protean II system. Proteins on the gel were stained with Coomassie Brilliant Blue. SOD activity was detected according to Beauchamp and Fridovich (19) after gel electrophoresis on 12% polyacrylamide gels. Staining was also performed in the presence of azide, cyanide, and hydrogen peroxide.

Activity Assay-- SOD activity was determined by the ferricytochrome c assay (20), where xanthine and xanthine oxidase was used as the source of superoxide radicals and cytochrome c as the indicating scavenger of the radical. The assay mixture (3.0 ml) contained 50 mM phosphate buffer (pH 7.8), 0.1 mM EDTA, 50 µM xanthine, 20 µM cytochrome c, and 50 µl of sample. The absorbance at 550 nm was monitored with a spectrophotometer after the addition of 50 µl of a solution containing 0.2 units/ml xanthine oxidase. One unit of SOD was defined as the amount of enzyme, which inhibits the rate of cytochrome c reduction by 50% at pH 7.8 and 25 °C, calculated from the initial rate of reaction. The concentration of the enzyme solution was determined spectrophotometrically with the calculated molar absorbance coefficient ₂₈₀ = 50,420 M¹ cm¹ per subunit (21).

Stopped-Flow Measurements-- To follow the reaction of superoxide and SOD we used the model SX-18MV stopped-flow spectrophotometer from Applied Photophysics equipped with a 1-cm observation cell at room temperature. Calculation of kinetic parameters from experimental traces was performed with the SpectraKinetic work station v4.38 interfaced to the instrument. The superoxide solution was prepared in a glove box; 133 mg of potassium superoxide and 800 mg of 18-crown-6-ether were dissolved in 6.7 ml of dimethyl sulfoxide (Me₂SO) and 3.3 ml of dimethylformamide (DMF) and filtered. Both aprotic organic solvents were dried over a 3-Å molecular sieve. The sequential mixing mode was utilized to follow the decrease of superoxide concentration. In the first step one part of superoxide solution was mixed with 10 parts of 5 mM buffer at pH 10. After a delay time of 10 ms, 10 parts of this mixture were combined with 10 parts of buffer at pH 7-10 with or without the enzyme. The following molar absorbance coefficient was used for recording of the superoxide decay (wavelength is shown in parentheses): 2250 M¹ cm¹ (245 nm) (22). Prior to data collection for superoxide decay, several shots of the buffer with Me₂SO/DMF mixture were used to determine the baseline. All stopped-flow determinations were measured in 90 mM of final buffer concentration at various pH values between pH 7 and 10, containing 100 µM EDTA, and at least three determinations were performed per substrate and enzyme concentration.

Sequence Analysis-- The amino acid sequences used in this work were extracted from the protein entries in the Entrez Protein search and retrieval system of NCBI (at http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?db = Protein), which includes data from Swiss-Prot, PIR, PRF, PDB, GenBank^TM, and RefSeq. Multiple sequence alignment was performed using ClustalX (23) and the following parameters: gap opening 10.0, gap extension 0.05, and the BLOSUM protein weight matrices. Phylogenetic analysis was performed using the PHYLIP package, version 3.57 (24).

A hydrophathy plot was carried out using WinPep version 3.0 (25) using the scale of Kyte and Doolittle (26). The size of sliding window was fixed to 11, and the hydropathy threshold for transmembrane helices was set at 1.6.

Secondary structure prediction for superoxide dismutase was performed using the program PSIPRED version 2.2 (27) at bioinf.cs.ucl.ac.uk/psipred/. It contains the subprograms GenTHREADER for recognizing the fold of a protein and MEMSAT 2 to infer the topology of transmembrane proteins.

To predict the localization site of the superoxide dismutase in cells the PSORT program, version 6.4 (28), was applied. It is available at psort.nibb.ac.jp/. The information of an amino acid sequence and its source origin, in this case Gram-negative bacteria, was used as input. The input sequence was analyzed by applying the stored rules for various sequence features of known protein sorting signals. Finally, the possibility for the input protein to be localized at each candidate site was reported.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Superoxide Dismutases of Anabaena PCC 7120-- The genome of Anabaena PCC 7120 was completely sequenced at the Kazusa DNA Research Institute (www.kazusa.or.jp/cyano/Anabaena/). Screening the genome shows that it possesses two potential genes that code for two distinct superoxide dismutases, namely one (sodA) coding for an iron-containing (FeSOD) and one (sodB) coding for a manganese-containing superoxide dismutase (MnSOD). To look for the localization of the corresponding active gene products, the cyanobacterium Anabaena PCC 7120 was grown under photoautotrophic conditions, and cells were harvested. French press extrusion produced whole cell extracts, and ultracentrifugation was used to separate crude membranes (cytoplasmic and thylakoid membranes) and cytoplasmic components. The membranes were solubilized by incubation in 2% dodecyl maltoside. All fractions were tested for superoxide dismutase activity and used for nondenaturating polyacrylamide gel electrophoresis combined with staining of SOD activity. Superoxide dismutase activity measured photometrically by the ferrocytochrome c assay can be related to the cytosolic fraction ((15.5 ± 2) units per mg of protein) and the membrane fraction ((3.1 ± 0.7) units per mg of protein). These specific activities are similar to that of SODs reported for other cyanobacteria (5) as well as for crude extracts of aerated E. coli cells (13.5 units/mg protein (29)), Bacillus subtilis (13.7 units/mg protein (30)) or rat liver (21 units/mg protein (31)). In addition, the determined ratio of membrane-bound SOD activity to cytosolic activity (1:5) is similar to that determined for MnSOD activity to FeSOD activity in P. boryanum (1:7) and Anabaena variabilis (1:6) (5).

Native PAGE clearly shows two proteins with SOD activity when the whole cell extract was separated on native PAGE, whereas one distinct band was seen when either the solubilized crude membrane fraction (i.e. both cytoplasmic and thylakoid membranes) (Fig. 1, lane 4) or the cytosolic fraction (Fig. 1, lane 5) were loaded on PAGE. Activity staining of cell extracts in the presence of high concentration of hydrogen peroxide demonstrated that the membrane-associated protein was still active, whereas the cytosolic SOD was inactive (not shown). This indicated that the membrane-associated SOD could be the manganese-containing enzyme. Addition of azide, but not cyanide inhibited both SOD activities (not shown).

View larger version (99K): [in this window] [in a new window]   Fig. 1.   Polyacrylamide gel electrophoresis of cell extracts. Lanes 1-3, SDS-PAGE; lane 1, molecular mass marker; lane 2, E. coli extract containing overexpressed MnSOD (without transmembrane segment); lane 3, E. coli extract containing overexpressed MnSOD (without transmembrane and linker segment); lanes 4-5, nondenaturing PAGE of cell extracts from Anabaena PCC 7120 stained for superoxide dismutase activity; lane 4, membrane extract (thylakoid and cytoplasmic membrane); lane 5, cytosolic extract.

Since a typical cyanobacterium comprises two types of bioenergetically competent membrane systems, viz. the chlorophyll-containing intracytoplasmic or thylakoid membrane (ICM) and the cytoplasmic or plasma membrane (CM) (32), CM and ICM were separated and purified by discontinuous sucrose density gradient centrifugation. Whereas cytoplasmic membranes gave an orange layer at 30% sucrose, thylakoid membranes form a dark green layer at 35/42% sucrose. Hydrogen peroxide-insensitive SOD activity could be assigned to both membrane fraction with 2.0 ± 0.7 units per mg of protein in ICM and 3.1 ± 0.5 units per mg of protein in CM, respectively. These results clearly show that the MnSOD is located in both cyanobacterial membranes in nearly equal concentrations. So it is tempting to speculate that there is no selective signal for targeting the protein to one of the two membranes.

Sequence Analysis and Secondary Structure Prediction-- To clarify the mode of CM and ICM association of MnSOD, the Anabaena MnSOD was analyzed and aligned with FeSODs and MnSODs known from other organisms. Fig. 2 depicts the nucleotide sequence of the sodB gene and the deduced amino acid sequence, including upstream and downstream elements. It shows a 810-bp open reading frame (ORF) from the start site at ATG to the termination codon TAA, coding for a polypeptide of 270 amino acids. Comparing the amino acid sequence with those from MnSODs where a high resolution x-ray structure is available (33-36) shows that Anabaena MnSOD contains an N-terminal extension consisting of eight residues containing positively charged amino acids followed by a hydrophobic patch of 17 amino acids and a linker region that precedes the intrinsic catalytic part (Fig. 3). A hydropathy plot unequivocally demonstrated that the primary sequence of Anabaena MnSOD comprises a membrane-spanning domain at the N terminus (not shown). Interestingly, this seems to be a general structural feature of all so far known cyanobacterial MnSODs with the exception of MnSOD2 from P. boryanum (Fig. 3).

View larger version (60K): [in this window] [in a new window]   Fig. 2.   Nucleotide sequence of the sodB gene of Anabaena PCC 7120 coding for the manganese-containing superoxide dismutase with upstream sequences and the deduced amino acid sequence. TS, transcription start site; RB, ribosome binding site. The ribosome binding sequence and start and stop codon are underlined; transcription start base and promoter bases in the 10 and 35 regions are boxed. Numbering starts at the first base of the coding sequence.

View larger version (116K): [in this window] [in a new window]   Fig. 3.   Comparison of FeSOD and MnSOD from various cyanobacterial species. The derived amino acid sequences are aligned using the program ClustalX, version 1.81. Amino acids at the N terminus of MnSODs, which may form a transmembrane helix, are in white with black background. Residues coordinating the metal center are boxed. Fe, F1-3 FeSODs; Mn, M1-3 MnSODs 1-3; A.7120, Anabaena PCC 7120; N.comm., Nostoc commune; N.linc., Nostoc linckia; N.punc., N. punctiforme; P.bory., P. boryanum; S.6301, Synechococcus PCC 6301; S.6803, Synechocystis PCC 6803; S.7942, Synechococcus PCC 7942.

Using the program MEMSAT of PSIPRED for topology analysis, it was predicted that residues from Phe-9 to Cys-25 form a central transmembrane helix segment and that residues 1-8 are located on the cytoplasmic side of the membrane, whereas most of the protein including the catalytic domains are positioned on the outside, in this case periplasm or the thylakoid lumen. This classifies it as a type 2 membrane protein. Until now, the cyanobacterial SODs are the only known manganese- or iron-containing superoxide dismutases that are membrane-anchored by a transmembrane helix. The essential catalytic part, which is connected with the transmembrane helix by a linker region (Fig. 3), is homologous to MnSODs and contains three conserved histidines and one aspartate that function as ligands for the metal ion (33-36). In contrast to the manganese enzymes, cyanobacterial FeSODs do not contain an N-terminal extension with a hydrophobic patch that is responsible for membrane insertion. These findings underline the different localizations of FeSOD and MnSOD in Anabaena PCC 7120.

Phylogenetic Analysis-- To establish the degree of evolutionary links among the various iron- and manganese-containing superoxide dismutases, the most likely tree was computed using the programs ClustalX and PROTDIST, FITCH, and CONSENSE from the PHYLIP package. The tree was based on the alignment of the entire amino acid sequences of the various SODs. The Dayhoff PAM matrix was used for calculation of the unrooted distance tree. Before the analysis the bootstrap resampling method was applied with 100 replicates. The output was subjected to the FITCH procedure and these output trees were further analyzed by the CONSENSE method. The program DrawTree was utilized for visualization of the most probable tree. For this analysis 83 protein sequences of iron- or manganese-containing SODs were selected including two bacterial cambialistic SODs, two FeSODs from Archaea, three fungal mitochondrial MnSODs, seven animal mitochondrial MnSODs, three plant mitochondrial MnSODs, three plant chloroplast FeSODs, six cyanobacterial MnSODs, and eight cyanobacterial FeSODs. All other enzymes are bacterial FeSODs or MnSODs (see Fig. 4). Two main clusters are easily discernable on the constructed tree. The FeSODs are normally well separated from the MnSODs but with several exceptions. Within the FeSODs the cambialistic SODs are grouped. This SOD can accept iron or manganese ions as cofactors in the active site, and in both cases, they possess substantial catalytic activity. They do not form a separated cluster but are spread throughout the FeSODs. What also is remarkable is that FeSODs from Archaea and thermophilic Bacteria form a clade within the MnSOD cluster (lower left part of Fig. 4). Freshwater cyanobacteria contain soluble FeSODs, which are grouped within a branch also containing FeSODs of plant chloroplasts (upper right part of Fig. 4). Some freshwater cyanobacteria additionally possess one or more membrane-bound MnSODs. Whereas in the completely sequenced unicellular cyanobacterium Synechocystis PCC 6803 only one FeSOD is present, the filamentous cyanobacteria also have encoded at least one MnSOD with high homology to each other. For example the homology for various cyanobacterial MnSODs are as follows (values of percent identity and percent similarity are given in parenthesis): Anabaena 7120-N. punctiforme M1 (57/71), Anabaena 7120-N. punctiforme M2 (68/78), Anabaena 7120-P. boryanum M1 (56/70); otherwise the homology between Anabaena 7120 and E. coli is 50/63, and that between Anabaena MnSOD and E. coli FeSOD is 44/59.

View larger version (26K): [in this window] [in a new window]   Fig. 4.   Suspected phylogenetic relationships of iron- and manganese-containing superoxide dismutases. The unrooted phylogenetic tree of SODs is the most likely one calculated with the programs ClustalX and PROTDIST, FITCH, and CONSENSE from the PHYLIP package. Branch length represents calculated phylogenetic distances. Cm, cambialistic SOD; Fe, F1-3, FeSODs; Mn, M1-3, MnSODs 1-3; A.7120, Anabaena PCC 7120; A.aeol., Aquifex aeolicus; A.fumi., Aspergillus fumigatus; A.pyro., Aquifex pyrophilus; A.thal., Arabidopsis thaliana; A.tume; Agrobacterium tumefaciens; B.bovi, Babesia bovis; B.burg., Borrelia burgdorferi; B.cald., Bacillus caldotenax; B.pseu., Burkholderia pseudomallei; B.stea., B. stearothermophilus; B.taur., Bos taurus; C.coli., Campylobacter coli; C.cres., Caulobacter crescentus; C.diph., Corynebacterium diphtheriae; C.eleg., Caenorhabditis elegans; C.muri, Chlamydia muridarum; C.pneu, Chlamydophila pneumoniae; C.sapi., Callinectes sapidus; D.radi., Deinococcus radiodurans; D.vulg., Desulfovibrio vulgaris; E.coli, E. coli; G.gall., Gallus gallus; G.poly, Gonyaulax polyedra; H.infl., Haemophilus influenzae; H.pylo., Helicobacter pylori; H.sapi., Homo sapiens; L.mono., Listeria monocytogenes; L.chag., Leishmania chagasi; L.pneu, Legionella pneumophila; M.avi., Mycobacterium avium; M.musc., Mus musculus; N.comm., N. commune; N.cras., Neurospora crassa; N.gono., Neisseria gonorrhoeae; N.linc., N. linckia; N.punc., N. punctiforme; P.aeru., Pseudomonas aeruginosa; P.bory., P. boryanum; P.freu., Propionibacterium freudenreichii; P.ging., Porphyromonas gingivalis; P.oval., Pseudomonas ovalis; P.sati., Pisum sativum; P.syri., Pseudomonas syringae; R.caps., Rhodobacter capsulatus; R.norv., Rattus norvegicus; R.prow., Rickettsia prowazekii; S.6301, Synechococcus PCC 6301, S.6803, Synechocystis PCC 6803; S.7942, Synechococcus PCC 7942; S.acid, Sulfolobus acidocaldarius; S.cere., Saccharomyces cerevisiae; S.solf., Sulfolobus solfataricus; S.aure., Staphylococcus aureus; S.pyog., Streptococcus pyogenes; S.typh., Salmonella typhimurium; S.meli., Sinorhizobium meliloti; T.acid., Thermoplasma acidophilum; T.cruz, Trypanosoma cruzi; T.foet., Trichomonas fetus; T.ther., Thermus thermophilus; T.vagi., Trichomonas vaginalis; V.chol., Vibrio cholerae; Y.pest., Yersinia pestis; Z.aeth., Zantedeschia aethiopica, and Z.mays., Zea mays.

Recombinant Protein-- To further characterize this membrane-bound MnSOD an attempt was made to overexpress and purify the enzyme. At first the whole sequence including the membrane-spanning segment (using primers 1a and 2) was inserted into the expression vector and transferred into E. coli. Screening of 50 clones showed that 43 contained the insert in the right order but searching of these 43 clones by SDS-PAGE for overexpressed protein did not give a positive result. This could be due to incorrect targeting or folding within E. coli cells and concomitant degradation because of the hydrophobic N-terminal extension. To bypass this problem two truncated forms of MnSOD were constructed. One did not contain the hydrophobic patch (amino acids 2-29 had been removed; primers 1b and 2) (MnSOD(28)) and the other additionally do not possess the linker region connecting the hydrophobic patch with the catalytic part (amino acids 2-61 have been removed; primers 1c and 2) (MnSOD(60)). In both cases most of the E. coli clones overexpressed the MnSOD in soluble form. The left part of Fig. 1 shows the soluble extracts of E. coli after gel electrophoresis. At lane 2 a strong band at 28 kDa corresponding to MnSOD(28) can be seen, and analogously at lane 3 a distinct band at 25 kDa is visible (MnSOD(60)). Both recombinant proteins contain a C-terminal hexahistidine tag and, therefore, were purified by metal chelate affinity chromatography and subsequent gel filtration. The yield in both cases was ~70 mg of highly purified SOD per liter of E. coli culture. The isoelectric points of MnSOD(60) and MnSOD(28) were determined to be at pH 6.7 and 6.9, which fits well with the calculated values of 6.3 and 6.4, respectively.

Physico-Chemical Characterization of Anabaena MnSOD-- The optical absorption spectrum shows a protein peak at 280 nm and characteristic absorbance for the manganese at 480 nm with an absorbance coefficient of ₄₈₀ = 910 M¹ cm¹ (22). From the spectrum a manganese content of 0.94 manganese per subunit was calculated (Fig. 5A). Addition of sodium dithionite removes the peak at 480 nm due to the reduction of Mn³⁺ to Mn²⁺. Gel filtration of both MnSODs on a calibrated Superdex 200 column yielded a single peak whose elution volume corresponded to an estimated molecular mass of (45 ± 5) kDa (MnSOD(60)) and (55 ± 5) kDa (MnSOD(28)), respectively, whereas SDS-PAGE gave a single band at 25 ± 2 kDa (MnSOD(60)) and (28 ± 2) kDa (MnSOD(28)), respectively. These data demonstrate that both constructs, like many other FeSODs or MnSODs, are homodimeric and that the linker region has no effect on the oligomerization state. Activity staining of a 12% native gel loaded with purified MnSOD showed a unique major band. The specific activity using the ferricytochrome assay gave values of (660 ± 90) units mg¹ (MnSOD(28)) and (650 ± 80) units mg¹ (MnSOD(60)) at pH 7.8, respectively.

View larger version (26K): [in this window] [in a new window]   Fig. 5.   Absorption spectrum and stopped-flow measurements of Anabaena MnSOD. A, UV-visible spectrum of 20 µM MnSOD. The inset shows the manganese-specific peak at 480 nm from a solution of 90 µM MnSOD. Both spectra were recorded in 100 mM phosphate buffer, pH 7.0. B, Stopped flow time traces at various MnSOD concentrations with 86 µM superoxide recorded at 245 nm and room temperature in 90 mM carbonate buffer, pH 10. C, pseudo-first-order rate constants between the MnSOD and superoxide. D, effect of pH value rate constants of the reaction of MnSOD and superoxide.

This assay was also performed in the presence of 10 mM inhibitor such as H₂O₂, NaCN, and NaN₃. Whereas H₂O₂ and NaCN were not able to inhibit the Anabaena MnSOD, it was inhibited at 10 mM NaN₃ by 55%. Also inhibition studies using the activity stain on native gels confirmed these results when azide and H₂O₂ where included in the developing solution. This is in agreement with inhibition studies of other SODs. In general MnSODs are inhibited by NaN₃ but not NaCN or H₂O₂, FeSODs are sensitive to NaN₃ and H₂O₂ but not to NaCN, and CuZnSODs are inhibited by H₂O₂ and NaCN. Titration of 100 µM Anabaena MnSOD in 100 mM phosphate buffer at pH 7.0 by increasing the NaN₃ concentration and recording the difference spectra gave a dissociation constant for azide of 0.84 mM.

Stopped-Flow Kinetics of Recombinant Anabaena MnSOD-- The catalysis of the decay of superoxide by both recombinant variants was also investigated by using the stopped-flow technique. Similar rates were obtained that underline that the catalytic domain has the full enzymatic capacity in superoxide dismutation. For superoxide preparation KO₂ was dissolved in DMF/Me₂SO including 18-crown-6-ether under anaerobic conditions in a glove box. To reduce the final concentration of organic solvent, the superoxide solution was mixed with 5 mM carbonate buffer at pH 10 in a 1:10 ratio, and after a delay time of 10 ms where only a minor fraction of superoxide decayed, it was mixed with various concentrations of SOD in a ratio of 1:1 in the appropriate buffer at pH 7-10. This ensures that the concentration of organic solvent is not higher than 5% (v/v) and accurate mixing of organic and aqueous solvent is guaranteed. Background first-order processes could be completely prevented and in the absence of SOD the curves show second-order self-dismutation of the superoxide radical. In Fig. 5B the upper line shows this self-dismutation at pH 10. Plotting the reciprocal values 1/[superoxide] versus time gives a perfect linear fit. Also the curves obtained at lower pH values (pH range of 7-10) exhibit no background first-order reactions induced by contaminants and show second-order decay. To study the superoxide decay catalyzed by the Anabaena MnSOD, its concentration was varied between 0.1 and 2 µM SOD. So there is at least an initial excess of 40 times of superoxide compared with the enzyme. At least three determinations of the pseudo-first-order rate constants, k_obs, were measured for each enzyme concentration, and the mean value was used to calculate the second-order rate constants. Plotting pseudo-first-order rate constants versus enzyme concentration gave excellent linear graphs (see Fig. 5C for pH 10) in the pH range 7-10. Taking into account the second-order self-dismutation under the conditions used in these experiments over the pH range 7-10, the slopes of the lines were used to calculate the second-order rate constants for the reaction of superoxide dismutase with superoxide. These second-order rate constants have a distinct pH dependence as can be seen in Fig. 5D. Increasing the pH results in a decrease of the rate constants, which is in agreement with other FeSODs and MnSODs but in contrast to CuZnSODs. What also can be seen from the plotting of pseudo-first-order rate constants versus enzyme concentration is that with decreasing pH values the intercept increases due to enhanced self-dismutation of superoxide.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Cyanobacteria are oxygenic phototrophic bacteria carrying out oxygenic photosynthesis and water-forming, O₂-reducing respiration in one and the same "noncompartmentalized" prokaryotic cell (37). Two types of morphologically more or less separate membrane systems occur in cyanobacteria, the chlorophyll-containing ICM and the chlorophyll-free CM. ICM and CM contain some identical electron transport components forming a dual functional photosynthetic-respiratory assembly in ICM but a pure respiratory chain in CM. Both ICM and CM surround individual and osmotically autonomous cellular compartments, viz. the thylakoid lumen and the cytosol, respectively. Fig. 6 shows a schematic presentation of the membrane-bound electron transport components of photosynthesis and respiration in cyanobacteria. Both bioenergetic processes permanently generate superoxide as a byproduct that has to be scavenged to prevent inactivation of e.g. enzymes of the Calvin cycle or the reaction centers of photosystems I and II. Fig. 6 includes the findings of this paper, which show that in filamentous cyanobacteria manganese superoxide dismutase is bound to both CM and ICM at the same extent with the catalytic portion of the protein being localized in the thylakoid lumen or the periplasm, whereas a soluble FeSOD is found in the cytosol. This different microcompartmentation of MnSOD and FeSOD guarantees that in Anabaena PCC 7120 all compartments contain SODs. This is important since cyanobacteria are exposed to extreme fluctuations of environmental conditions (e.g. light intensity or temperature) that affect the photon-utilizing capacity and hence the balance between oxygenic photosynthesis and aerobic respiration, the latter being strongly enhanced under many stress conditions (38).

View larger version (29K): [in this window] [in a new window]   Fig. 6.   Scheme of bioenergetic membrane functions in a cyanobacterium (42) including localization of superoxide dismutases. CM, plasma membrane; ICM, thylakoid membrane; PSI, PSII, photosystems I and II; PQ, plastoquinone; fd, ferredoxin; FNR, ferredoxin-NADP reductase; c6, soluble cytochrome c554; PC, plastocyanin; DH1/2, either bacteria-like nonproton-translocating one-subunit NADH dehydrogenase or mitochondria-like multisubunit NADH dehydrogenase; Cyt bc, cytochrome bc complex; Cyt aa3, cytochrome c oxidase; F-type and P-type, F-type and P-type H+-translocating ATPases; AP, proton-sodium antiporter; FeSOD and MnSOD; iron- and manganese-containing superoxide dismutase.

In this regard cyanobacteria are unique. MnSODs from other organisms that are described so far are soluble proteins located either in the cytosol of bacteria or the matrix of mitochondria (33). The cyanobacterial MnSODs are highly homologous and form a discernible cluster on the calculated phylogenetic tree shown in Fig. 4.

The two recombinant variants MnSOD(28) and MnSOD(60), which lack either the hydrophobic region or both the hydrophobic and the linker region, are both homodimeric proteins exhibiting the full catalytic activity underlining that (i) the linker region is not involved in oligomerization and that (ii) the soluble catalytic portion is fully active in superoxide dismutation as demonstrated by both conventional and stopped-flow spectroscopy. The sequence alignment of Fig. 3 clearly suggests, on the basis of the known structures of MnSODs (33-36), that the catalytic portion of Anabaena MnSOD is also a two-domain protein whose active site is located at the junction of a N-terminal -helical domain and a C-terminal / domain and whose domains contribute ligands for the manganese center (Fig. 3). All of the so far published structures show an extended hydrogen bond network controlling the activity of the enzyme and connecting the active site of one monomer with amino acid residues from the second polypeptide chain. Based on the sequence alignments (Fig. 3) and the known structures of the homodimeric proteins from Bacillus stearothermophilus (35) and E. coli (33) it is reasonable to assume that a similar hydrogen bond network is present in Anabaena MnSOD.

The visible absorption spectra of both recombinant proteins have a maximum at 480 nm, which is pH-dependent (not shown) and disappears upon reduction as do the enzymes from other bacterial sources. The specific activity is lower than that described for other species. This was also seen utilizing stopped-flow spectrometry to follow directly the decay of superoxide in the presence of Anabaena MnSOD. Normally the catalysis of the decay of superoxide in the presence of MnSOD shows a biphasic pattern in which a first-order decay of superoxide is quickly followed by an extended region of a slower disappearance of superoxide. This was interpreted by the formation of an inhibited intermediate during catalysis. The inactive state has been observed spectrophotometrically and has been postulated to contain a side-on complex of Mn(III) and peroxide (39, 40). Active enzyme is regenerated by dissociation of this complex to the Mn(III) and free peroxide, and it is thought that this regeneration of active enzyme dominates the kinetics of the second slower phase of the catalysis (39). Based on the observation that with the human MnSOD the first rapid phase occurred within a few milliseconds and could be observed only with pulse radiolysis (39), the human protein has been shown to be much more susceptible to this reversible inactivation than bacterial MnSODs. However, the two investigated recombinant proteins MnSOD(28) and MnSOD(60) did not show a deviation from first-order kinetics of superoxide decay in the presence of enzyme at broad concentration range thus allowing a simple kinetic analysis as described by Riley et al. (41). This suggests that the reaction product hydrogen peroxide does not have an important impact on catalysis of Anabaena MnSOD. Several observations support this. (i) A fast phase could not be monitored using stopped-flow spectroscopy. This could mean that the Anabaena protein exhibits a similar susceptibility to product inhibition as the human enzyme or that the presence of hydrogen peroxide has no impact on SOD catalysis. (ii) The [O]/[MnSOD] ratio in our experiments varied from 40 to 600, however the shape of time traces did not change. In all cases k_obs values could be obtained by fitting the time traces to a single exponential equation. These pseudo-first-order rate constants were strongly dependent on the enzyme. The resulting plot was linear over the entire concentration range employed confirming that the reaction is first-order in enzyme concentration, thus allowing the determination of actual bimolecular rate constants. (iii) The fact that these rates increased by decreasing pH values strongly suggests that an inhibitory intermediate plays a minor role in superoxide dismutation mediated by Anabaena MnSOD. This is based on the data about human MnSOD where the rate constant for the slower phase (determined by formation and dissociation of the inhibitory complex) has been shown to be not affected by pH values in contrast to the preceding fast phase where the rates increased by decreasing pH values (39). (iv) And finally, both the activity staining on native PAGE and the steady-state kinetics showed a negligible influence of H₂O₂ even at a millimolar concentration. Crystallization studies of both MnSOD(28) and MnSOD(60) are in progress to further analyze the differences in kinetics between cyanobacterial membrane-bound and other bacterial cytosolic MnSODs.

	FOOTNOTES

^* This work was supported by the Austrian Science Fund (FWF-Project P13069-CHE).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^** To whom correspondence should be addressed. Fax: 43-1-36006-6059; E-mail: cobinger@edv2.boku.ac.at.

Published, JBC Papers in Press, September 4, 2002, DOI 10.1074/jbc.M207691200

	ABBREVIATIONS

The abbreviations used are: SOD, superoxide dismutase; DMF, dimethylformamide; CM, cytoplasmic or plasma membrane; ICM, intracytoplasmic or thylakoid membrane; ORF, open reading frame; PCC, Pasteur Culture Collection.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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