Biochemical Characterization of a Membrane-bound Manganese-containing Superoxide Dismutase from the CyanobacteriumAnabaena PCC 7120 (2002)

The filamentous cyanobacteriumAnabaena 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(Δ28)) or the hydrophobic and the linker region (MnSOD(Δ60)) 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 mg− 1The 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 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(Δ28)) or (MnSOD(Δ60)) concentrations is first-order in enzyme concentration allowing the calculation of catalytic rate constants which increase with decreasing pH: 8 × 106m− 1The 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 Collections− 1The 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 (pH 10) and 6 × 107m− 1The 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 s− 1The 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(pH 7). The physiological relevance of these findings is discussed with respect to the bioenergetic peculiarities of cyanobacteria. The filamentous cyanobacteriumAnabaena 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(Δ28)) or the hydrophobic and the linker region (MnSOD(Δ60)) 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 mg− 1The 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 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(Δ28)) or (MnSOD(Δ60)) concentrations is first-order in enzyme concentration allowing the calculation of catalytic rate constants which increase with decreasing pH: 8 × 106m− 1The 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 Collections− 1The 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 (pH 10) and 6 × 107m− 1The 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 s− 1The 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(pH 7). The physiological relevance of these findings is discussed with respect to the bioenergetic peculiarities of cyanobacteria. 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 ( 1The 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 O2) 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 (1Johnson E.A. Schroeder W.A. Adv. Biochem. Eng. Biotechnol. 1996; 53: 119-178PubMed Google Scholar). The second pathway of molecular oxygen activation results in the formation of the superoxide radical ( O2−⋅) by interaction with electron acceptors of photosystem I or reduced ferredoxin (2Asada K. Foyer C.H. Mullineaux Causes of Photooxidative Stress in Plants and Amelioration of Defense Systems. CRC Press, Boca Raton1994: 77-104Google Scholar). 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) (2Asada K. Foyer C.H. Mullineaux Causes of Photooxidative Stress in Plants and Amelioration of Defense Systems. CRC Press, Boca Raton1994: 77-104Google Scholar). 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) 1The 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 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 (3Asada K. Yoshikawa K. Takahashi M. Maeda Y. Enmanji K. J. Biol. 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Wright D.J. Wilmoth G. Prickett T.F. Helm R.F. Gregory E.M. Potts M. J. Bacteriol. 2000; 182: 189-197Crossref PubMed Scopus (131) Google Scholar, 11Laudenbach D.E. Trick C.G. Straus N.A. Mol. Gen. Genet. 1989; 216: 455-461Crossref PubMed Scopus (28) Google Scholar, 12Campbell W.S. Laudenbach D.E. J. Bacteriol. 1995; 177: 964-972Crossref PubMed Google Scholar, 13Liu Y. Zhou R. Zhao J. Biochim. Biophys. Acta. 2000; 1491: 248-252Crossref PubMed Scopus (8) Google Scholar). These reports as well as genome analysis (www.kazusa.or.jp/cyano/) show that cyanobacteria possess either one or more SOD genes. The unicellular cyanobacteriumSynechocystis PCC 6803 contains a single FeSOD contains one FeSOD as well as three MnSOD genes W.S. Laudenbach D.E. J. Bacteriol. 1995; 177: 964-972Crossref PubMed Google and Nostoc contains one FeSOD and two MnSOD genes. The reports the physiological of cyanobacterial that these during or from the of In Nostoc a for an FeSOD in to (10Shirkey B. Kovarcik D.P. Wright D.J. Wilmoth G. Prickett T.F. Helm R.F. Gregory E.M. Potts M. J. Bacteriol. 2000; 182: 189-197Crossref PubMed Scopus (131) Google Scholar). the cyanobacteriumAnabaena PCC 7120 (now renamed Nostoc PCC 7120) as a to the for superoxide dismutase shown that both membrane and SOD of the genome PCC 7120 revealed that are two genes for one a high to iron-containing and the other to manganese-containing superoxide dismutase In this localization of the MnSOD in both cytoplasmic and thylakoid It is shown MnSOD is unique in being a membrane-bound is reported as well as and the of rate constants at were from the molecular molecular and superoxide from the DNA and and from the from from DNA from and DNA from other were of of Anabaena PCC 7120 were in R. M. G. Bacteriol. PubMed Google at for and with containing of were by × with and in the same to a of The to and at for and × the in containing for through a at and and The used to membranes and by × or to with for This solution with and in and × the by were to and and G.A. M. M. 1989; PubMed Scopus Google Scholar). DNA and protein sequence of the manganese-containing superoxide dismutase from the the genome for Anabaena PCC at The were and from of these three an and an and with a a sequence for a and a One of the and from Anabaena as the and the DNA were used for the for at for for and for and at for The a and the DNA and using the DNA and This DNA with the and and into and PubMed Scopus Google Scholar). The by the S. S. A. PubMed Scopus Google Scholar). with the by the recombinant were and an at in containing and of the with containing the same and of superoxide dismutase by the of of at cells were by × and at in containing and and were and the cells were by with The and from the the superoxide dismutase The to and and a × with and with containing and at The with of the and proteins were with of a of in containing The proteins were and the with were a with containing at were using molecular the with the in the range of as by PubMed Scopus Google using and the the were with SOD activity to and Biochem. PubMed Scopus Google also in the presence of and hydrogen SOD activity by the ferricytochrome assay J. Biol. Chem. Full Text PDF PubMed Google and used as the of superoxide and as the of the The assay (pH and of The at with a the of of a solution containing One unit of SOD as the of which the rate of by at and from the rate of The concentration of the enzyme solution with the 1The 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 1The 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 per G. T. 1995; PubMed Scopus Google Scholar). To the reaction of superoxide and SOD used the stopped-flow from with a at of from with the to the The superoxide solution in a of superoxide and of were in of and of and were a molecular The to the of superoxide In the one of superoxide solution with of at a of of this were with of at with or the The used for of the superoxide decay is shown in 1The 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 1The 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 J. Chem. Scopus Google Scholar). to for superoxide several of the with were used to the stopped-flow were in of concentration at various and containing and at three were per and enzyme The sequences used in this were from the protein in the and of which from and sequence using M. M. Biochem. 1998; 23: Full Text Full Text PDF PubMed Scopus Google and the and the protein analysis using the J. 1996; PubMed Google Scholar). A using PubMed Scopus Google using the of and J. R.F. J. Mol. Biol. PubMed Scopus Google Scholar). The of to and the for transmembrane at for superoxide dismutase using the J. Mol. Biol. PubMed Scopus Google at It contains the for the of a protein and to the of transmembrane To the localization of the superoxide dismutase in cells the K. P. Biochem. Full Text Full Text PDF PubMed Scopus Google It is at The of an sequence and in this Gram-negative used as The sequence by the for various sequence of known protein the for the protein to be localized at each The genome PCC 7120 at the DNA the genome that it possesses two genes that for two superoxide one for an iron-containing (FeSOD) and one for a manganese-containing superoxide dismutase (MnSOD). To for the localization of the active the Anabaena PCC 7120 and cells were produced and used to membranes and thylakoid and cytoplasmic The membranes were by in were for superoxide dismutase activity and used for with of SOD dismutase activity by the assay can be to the ± per of and the membrane ± per of These specific activities are similar to that of reported for other cyanobacteria S. Kanematsu S. Asada K. FEBS Lett. 1979; 103: 106-110Crossref Scopus (49) Google as well as for of E. cells protein E.M. J. Bacteriol. PubMed Google protein E.M. J. Bacteriol. PubMed Google or protein J. Biochem. PubMed Scopus Google In the of membrane-bound SOD activity to activity is similar to that for MnSOD activity to FeSOD activity in P. S. Kanematsu S. Asada K. FEBS Lett. 1979; 103: 106-110Crossref Scopus (49) Google Scholar). clearly two proteins with SOD activity the whereas one either the membrane both cytoplasmic and thylakoid or the were of in the presence of high concentration of hydrogen peroxide that the protein whereas the SOD This that the SOD be the manganese-containing of but not both SOD activities a two types of membrane the intracytoplasmic or thylakoid membrane and the cytoplasmic or plasma membrane M. G.A. Microbiol. Scopus Google and were and by Whereas cytoplasmic membranes an at thylakoid membranes form a green at SOD activity be to both membrane with ± per of protein in and ± per of protein in CM, These results clearly show that the MnSOD is in both cyanobacterial membranes in nearly it is to that is no for the protein to one of the two To the of and of MnSOD and with FeSODs and MnSODs known from other organisms. the sequence of the and the and It a open reading from the at to the for a of the sequence with from MnSODs a high is 1992; Full Text PDF PubMed Scopus Google Scholar, J. Mol. Biol. 1991; PubMed Scopus Google Scholar, J. Mol. Biol. 1998; Scopus Google Scholar, J. Biol. Chem. 1998; Scopus Google that Anabaena MnSOD contains an of containing acids by a hydrophobic of acids and a linker region that the catalytic A that the sequence of Anabaena MnSOD a at the this to be a of known cyanobacterial MnSODs with the of of FeSOD and MnSOD from various cyanobacterial The sequences are using the acids at the of which form a transmembrane helix, are with the metal are MnSODs PCC Nostoc Nostoc PCC PCC PCC Using the of for it that from to form a transmembrane and that are the cytoplasmic of the whereas of the protein the catalytic domains are positioned the in this periplasm or the thylakoid lumen. This it as a membrane the cyanobacterial are the known or iron-containing superoxide dismutases that are by a transmembrane The catalytic which is with the transmembrane by a linker region is homologous to MnSODs and contains three and one that as for the metal ion 1992; Full Text PDF PubMed Scopus Google Scholar, J. Mol. Biol. 1991; PubMed Scopus Google Scholar, J. 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The of and were to be at and which well with the of and The a protein peak at and characteristic for the manganese at 480 with an of 1The 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 1The 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 J. Chem. Scopus Google Scholar). the a manganese of manganese per of the peak at 480 to the of to of both MnSODs a a single peak to an molecular of ± (MnSOD(Δ60)) and ± whereas a single at ± (MnSOD(Δ60)) and ± These that both other FeSODs or are homodimeric and that the linker region no the of a with MnSOD showed a unique The specific activity using the ferricytochrome assay of (660 ± 90) mg− 1The 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 (MnSOD(Δ28)) and ± mg− 1The 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 (MnSOD(Δ60)) at This assay also in the presence of such as and Whereas and were not able to the Anabaena it at by using the activity these results azide and in the This is in with of other In MnSODs are by but not or FeSODs are to but not to and are by and of Anabaena MnSOD in at by the and the a dissociation constant for azide of mm. 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These rate constants a as can be in the results in a of the rate which is in with other FeSODs and MnSODs but in to also can be from the of rate constants enzyme concentration is that with decreasing the to of superoxide. are oxygenic phototrophic bacteria oxygenic photosynthesis and in one and the same G.A. Biochem. 1984; PubMed Scopus Google Scholar). Two types of more or membrane in the and the and electron a in but a in and and the thylakoid and the 6 a of the membrane-bound electron of photosynthesis and in cyanobacteria. bioenergetic processes superoxide as a that to be scavenged to prevent inactivation of of the or the reaction of I and the findings of this which show that in filamentous cyanobacteria manganese superoxide dismutase is to both and at the same extent with the catalytic of the protein being localized in the thylakoid or the whereas a soluble FeSOD is in the This different of MnSOD and FeSOD that in Anabaena PCC 7120 This is cyanobacteria are to of (e.g. or that the and the oxygenic photosynthesis and the being G.A. S. B. FEMS Microbiol. Lett. Scopus Google Scholar). In this cyanobacteria are MnSODs from other organisms that are are soluble proteins either in the of bacteria or the of 1992; Full Text PDF PubMed Scopus Google Scholar). The cyanobacterial MnSODs are highly homologous and form a the phylogenetic shown in The two recombinant variants and which either the hydrophobic region or both the hydrophobic and the linker region, are both homodimeric proteins the catalytic activity that the linker region is not in and that the soluble catalytic is active in superoxide as by both and stopped-flow The sequence of clearly the of the known of MnSODs 1992; Full Text PDF PubMed Scopus Google Scholar, J. Mol. Biol. 1991; PubMed Scopus Google Scholar, J. Mol. Biol. 1998; Scopus Google Scholar, J. Biol. Chem. 1998; Scopus Google that the catalytic MnSOD is also a protein active is at the of a and a and domains for the manganese of the show an hydrogen the activity of the enzyme and the active of one with from the second the sequence and the known of the homodimeric proteins from J. Mol. Biol. 1998; Scopus Google and E. 1992; Full Text PDF PubMed Scopus Google it is to that a similar hydrogen is in Anabaena The of both recombinant proteins a at 480 nm, which is and as the from other The specific activity is lower than that for other This also stopped-flow to the decay of superoxide in the presence the of the decay of superoxide in the presence of MnSOD a in which a first-order decay of superoxide is by an region of a of superoxide. This by the formation of an during The and to a of and peroxide J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google T. J. Chem. 1991; Scopus Google Scholar). enzyme is by dissociation of this to the and and it is that this of active enzyme the of the second of the J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). the that with the MnSOD the a and be with J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google the protein shown to be more to this inactivation than the two recombinant proteins and not show a from first-order of superoxide decay in the presence of enzyme at concentration range allowing a analysis as by D.P. Biochem. 1991; PubMed Scopus Google Scholar). This that the reaction hydrogen peroxide not an of Anabaena A not be using stopped-flow This that the Anabaena protein a similar to as the enzyme or that the presence of hydrogen peroxide no SOD The in experiments from to the of not In be by the to a single These rate constants were the The the concentration range that the reaction is first-order in enzyme allowing the of rate The that these by decreasing that an a in superoxide by This is the MnSOD the rate constant for the by formation and dissociation of the shown to be not by in to the the by decreasing J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). both the activity and the showed a of at a of both and are in to the in cyanobacterial membrane-bound and other