Iron-containing Superoxide Dismutase Research Articles

The role of PAP4/FSD3 and PAP9/FSD2 in heat stress responses of chloroplast genes

Chloroplasts’ mechanisms of adaptation to elevated temperatures are largely determined by the gene expression of the plastid transcription apparatus. Gene disruption of iron-containing superoxide dismutase PAP4/FSD3 and PAP9/FSD2, which are parts of the DNA–RNA polymerase complex of plastids, contributed to a decrease in resistance to oxidative stress caused by the prolonged action of elevated temperatures (5 days, 30 °C). Under heat stress conditions, pap4/fsd3 and pap9/fsd2 mutants showed a decline in chlorophyll content and photosynthesis level, as measured by photosynthetic parameters, and a different amplitude of HSP gene response to heat stress. The expression of nuclear- and plastid-encoded photosynthesis genes and corresponding proteins was strongly inhibited in the mutants as compared with wild-type plants and was further suppressed or displayed no additional changes at 30 °C. NEP-dependent plastid genes, as well as NEP genes RPOTp and RPOTmp, were also downregulated in the mutants by high temperature or remained stable, unlike in wild-type seedlings where these genes were strongly upregulated. The results obtained correspond to the concept of the complex effect of various forms of reactive oxygen species under all types of stresses, including heat stress, and confirm the hypothesis of a new regulatory function in plastid transcription acquired by enzymatic proteins during evolution.

Detection and Sequencing of Iron Superoxide Dismutase Gene in Entamoeba histolytica Isolated from Patients with Diarrhea in Iraq.

Parasites are among serious health problems that threaten the public health of humans. Entamoeba histolytica is considered the second or third most common cause of death among parasitic diseases. The present study was conducted to determine the molecular detection and sequencing of iron-containing superoxide dismutase (FeSOD) genes of E. histolytica. This cross-sectional study was carried out within March-August 2020. Genomic DNA from stool samples was extracted using an AccuPrep® stool DNA Extraction Kit. Afterward, four DNA samples were subjected to sequence alignment analysis of the FeSOD gene in local E. histolytica human isolated using a Clustal W alignment tool (Mega 6.0).The iron-containing superoxide dismutase gene was present at 62.8% in 15 out of 24 E. histolytica samples. The results of sequence alignment analysis of the FeSOD gene in local E.histolytica human isolates indicated the number of mutations in each of the 4 isolates. Accordingly, there were 2 (16.66%) silent mutations and 1(6.66%) nonsense mutation. There are a variety of genetic strains of E. histolytica associated with diarrheal illness in Iraqi patients, which are unique to this country.

Identification of potential inhibitors of Trichomonas vaginalis iron-containing superoxide dismutase by computer-aided drug design approach

Trichomonas vaginalis is a human protozoan parasite that causes trichomoniasis, a common sexually transmitted disease. Metronidazole is a commonly used drug for the treatment of T. vaginalis infections. However, it was reported that the parasite has developed resistance to this drug. Therefore, a necessity of discovering new drugs that have different modes of action against T. vaginalis has emerged. In this computational study, T. vaginalis iron-containing superoxide dismutase (TvSOD) was selected as target protein because of its vital role in converting superoxide to oxygen and hydrogen peroxide and protecting the parasite against toxic reactive oxygen species (ROS). TvSOD was modeled using two different protein modeling programs, MODELLER and SWISS-MODEL, and then, small drug-like chemicals were screened for interaction with three different druggable pockets of the enzyme. The best interacting chemicals were then evaluated through molecular dynamics simulations (MDSs) for ligand stability. As a result, ligand-129817054 (7-(6-amino-1,2,3,4,5-pentahydroxyhexyl)-4-methylchromen-2-one) was determined to be a viable drug candidate based on docking scores and MDS results. Additional in vitro inhibition studies are necessary for the evaluation and assessment of the compound of interest as an effective TvSOD inhibitor.

In Vivo Biological Evaluation of a Synthetic Royleanone Derivative as a Promising Fast-Acting Trypanocidal Agent by Inducing Mitochondrial-Dependent Necrosis.

The life-long and life-threatening Chagas disease is one of the most neglected tropical diseases caused by the protozoan parasite Trypanosoma cruzi. It is a major public health problem in Latin America, as six to seven million people are infected, being the principal cause of mortality in many endemic regions. Moreover, Chagas disease has become widespread due to migrant populations. Additionally, there are no vaccines nor effective treatments to fight the disease because of its long-term nature and complex pathology. Therefore, these facts emphasize how crucial the international effort for the development of new treatments against Chagas disease is. Here, we present the in vitro and in vivo trypanocidal activity of some oxygenated abietane diterpenoids and related compounds. The 1,4-benzoquinone 15, not yet reported, was identified as a fast-acting trypanocidal drug with efficacy against different strains in vitro and higher activity and lower toxicity than benznidazole in both phases of murine Chagas disease. The mode of action was also evaluated, suggesting that quinone 15 kills T. cruzi by inducing mitochondrion-dependent necrosis through a bioenergetics collapse caused by a mitochondrial membrane depolarization and iron-containing superoxide dismutase inhibition. Therefore, the abietane 1,4-benzoquinone 15 can be considered as a new candidate molecule for the development of an appropriate and commercially accessible anti-Chagas drug.

Cytotoxic Activity of LCS-1 is not Only due to Inhibition of SOD1.

The cytotoxic activity of the pyridazin-3-one derivative LCS-1 was previously suggested to be due to the inhibition of superoxide dismutase 1 (SOD1). However, no direct evidence was provided that LCS-1 inhibits SOD1 within cells. In this study, we investigated the cytotoxic activity of LCS-1 against bloodstream forms of Trypanosoma brucei, a protozoan parasite that does not express copper/zinc-containing SOD1, but an iron-containing superoxide dismutase (FeSOD). At 250 µM, LCS-1 did not inhibit the activity of FeSOD in cell lysates of bloodstream forms of T. brucei, confirming that the compound is a specific inhibitor of SOD1. However, LCS-1 displayed substantial trypanocidal activity with a minimum inhibitory concentration of 10 µM and a half-maximal effective concentration of 1.36 µM, indicating that the cytotoxic action of the compound cannot solely be due to inhibition of SOD1. The results of this study is an important finding as it shows that LCS-1 has more than one cytotoxic mode of action.

Trypanothione Reductase and Superoxide Dismutase as Current Drug Targets for Trypanosoma cruzi: An Overview of Compounds with Activity against Chagas Disease.

It has been over a century since Carlos Chagas discovered the Trypanosoma cruzi (T. cruzi) as the causative agent of Chagas disease (CD), a neglected tropical disease with several socioeconomic, epidemiological and human health repercussions. Currently, there are only two commercialized drugs to treat CD in acute phase, nifurtimox and benznidazol, with several adverse side effects. Thus, new orally available and safe drugs for this parasitic infection are urgently required. One strategy of great importance in new drug discovery programmes is based on the search of molecules enabling to interfere with enzymes involved in T. cruzi metabolism. This review will focus on two of the most promising targets for the therapy of CD: trypanothione reductase (TR) and the iron-containing superoxide dismutase (Fe- SOD), which protect the parasite against oxidative damage by reactive oxygen species. A brief comparison of the function, mechanism of action and the active sites between T. cruzi TR and Fe-SOD with their analogues enzymes in human, glutathione reductase (GR) and the corresponding SODs, will be discussed. This review will also summarize the recent development and structure-activity relationships of novel compounds reported for their ability to selectively inhibit these targets, aiming to define molecular bases in the search for new effective treatment of CD.

Molecular Cloning and Biochemical Characterization of the Iron Superoxide Dismutase from the Cyanobacterium Nostoc punctiforme ATCC 29133 and Its Response to Methyl Viologen-Induced Oxidative Stress.

Superoxide dismutase (SOD) detoxifies cell-toxic superoxide radicals and constitutes an important component of antioxidant machinery in aerobic organisms, including cyanobacteria. The iron-containing SOD (SodB) is one of the most abundant soluble proteins in the cytosol of the nitrogen-fixing cyanobacterium Nostoc punctiforme ATCC 29133, and therefore, we investigated its biochemical properties and response to oxidative stress. The putative SodB-encoding open reading frame Npun_R6491 was cloned and overexpressed in Escherichia coli as a C-terminally hexahistidine-tagged protein. The purified recombinant protein had a SodB specific activity of 2560±48 U/mg protein at pH 7.8 and was highly thermostable. The presence of a characteristic iron absorption peak at 350nm, and its sensitivity to H2O2 and azide, confirmed that the SodB is an iron-containing SOD. Transcript level of SodB in nitrogen-fixing cultures of N. punctiforme decreased considerably (threefold) after exposure to an oxidative stress-generating herbicide methyl viologen for 4 h. Furthermore, in-gel SOD activity analysis of such cultures grown at increasing concentrations of methyl viologen also showed a loss of SodB activity. These results suggest that SodB is not the primary scavenger of superoxide radicals induced by methyl viologen in N. punctiforme.

Characterization of the iron-containing superoxide dismutase and its response to stress in cyanobacterium Spirulina (Arthrospira) platensis

Superoxide dismutase (SOD) is considered a primary antioxidant which defends against reactive oxygen species that are induced by environmental stress. In this study, we examined changes in SOD activity and expression in the cyanobacterium Spirulina (Arthrospira) platensis under iron and salinity stress; we characterized its induction under these stress conditions and we overexpressed the enzyme in a bacterial host for preliminary characterization. Analysis of SOD isoforms concludes that S. platensis was found to regulate only the iron-containing SOD isoform (FeSOD) in response to two types of stress that were tested. The FeSOD expression (on the level of both mRNA and enzyme activity) was induced by the stress conditions of salinity and iron levels. The FeSOD from S. platensis was overexpressed in Escherichia coli BL21. The recombinant FeSOD protein (about 23 kDa) was purified for characterization. It showed high specific activity and pH stability at 6.0–9.0, and it is relatively thermostable, retaining 45 % of its activity after 30 min at 90 °C. Phylogenetic analysis reveals that S. platensis FeSOD is grouped with the FeSODs from other cyanobacterial species and separated from those of the eukaryotic Chlorophyta, suggesting that the FeSOD gene may be used as a molecular marker in physiological, phylogenetic, and taxonomic studies. This study also suggests that the increased activity and expression of SOD may play a role in algal survival under stress conditions.

Expression and characterization of an iron-containing superoxide dismutase from Burkholderia pseudomallei

A superoxide dismutase (SOD) gene from Burkholderia pseudomallei, the causative agent of melioidosis, was cloned and expressed in Escherichia coli, and its product was functionally and physically characterized. The gene has an open-reading frame of 579 bp. The deduced amino acid sequence has 192 residues with a calculated molecular mass of ~22 kDa. Sequence comparison with other bacterial SODs showed that the protein contains typical metal-binding motifs and other Fe-SOD-conserved residues. The sequence has substantial similarity with other bacterial Fe-SOD sequences. The enzymatic activity of the expressed protein was inhibited by hydrogen peroxide but not by sodium azide or potassium cyanide, attributes that indeed are characteristic of typical bacterial Fe-SODs. Western blotting with antiserum against the recombinant Fe-SOD revealed that it is expressed in B. pseudomallei. Transformed E. coli that expressed the Fe-SOD had significantly increased SOD activity and was highly tolerant to paraquat-mediated replication inhibition, compared to transformed cells carrying an empty vector. Our results provide a basis for further biochemical characterization of the enzyme and elucidation of its role in the pathogenesis of B. pseudomallei.

Functional expression and characterization of an iron-containing superoxide dismutase of Acanthamoeba castellanii

Acanthamoeba spp. are free-living amoebae, but opportunistic infections of some strains of the organisms cause severe diseases such as acanthamoebic keratitis, pneumonitis, and granulomatous amoebic encephalitis in human. In this study, we identified a gene encoding iron superoxide dismutase of Acanthamoeba castellanii (AcFe-SOD) and characterized biochemical and functional properties of the recombinant enzyme. Multiple sequence alignment of the deduced amino acid sequence of AcFe-SOD with those of previously reported iron-containing SODs (Fe-SODs) from other protozoan parasites showed that AcFe-SOD shared common metal-binding residues and motifs that are conserved in Fe-SODs. The genomic length of the AcFe-SOD gene was 926 bp consisting of five exons interrupted by four introns. The recombinant AcFe-SOD showed similar biochemical characteristics with its native enzyme and shared typical biochemical properties with other characterized Fe-SODs, including molecular structure, broad pH optimum, and sensitivity to hydrogen peroxide. Immunolocalization analysis revealed that the enzyme localized in the cytosol of the trophozoites. Activity and expression level of the enzyme were significantly increased under oxidative stressed conditions. These results collectively suggest that AcFe-SOD may play essential roles in the survival of the parasite not only by protecting itself from endogenous oxidative stress but also by detoxifying oxidative killing of the parasite by host immune effector cells.

Purification and Partial Properties of a Superoxide Dismutase from Agave Hybrid No.11648

AbstractA superoxide dismutase enzyme was extracted from the leaves of Agave Hybrid No.11648 using homogenisation, ammonium sulphate fractional precipitation, diethylaminoethyl cellulose ion exchange and Sephadex gel filtration chromatography. A superoxide dismutase parallelogram‐shaped crystal was formed after packing at 4 °C for two days. The optimum pH was 8, and the optimum temperature was 40 °C. The crystal was relatively stable at temperatures below 60 °C and at pH values from 5 to 10. The subunit size was determined to be approximately 27.5 kDa by sodium dodecyl sulphate‐polyacrylamide gel electrophoresis. It was also demonstrated that the enzyme is an iron‐containing superoxide dismutase based on its sensitivity to inhibitors of potassium cyanide, hydrogen peroxide and chloroform‐ethanol and based on the results of ultraviolet absorption spectroscopy.

Cloning and expression of isoenzymes of superoxide dismutase in Haematococcus pluvialis (Chlorophyceae) under oxidative stress

Increasing evidence indicates that the biosynthesis and accumulation of the keto-carotenoid astaxanthin represents a chronic (in days or weeks) molecular defense mechanism in the green alga, Haematococcus pluvialis, to protect cells from abiotic stress. However, information with regard to the acute (in minutes or hours) response of the cells to stress is scarce. In this study, two cDNAs encoding manganese superoxide dismutase (MnSOD) were cloned and their full length nucleotide sequences were obtained. Quantitative real-time RT-PCR analysis revealed a general increase in MnSOD transcriptions in response to various abiotic stressors, e.g., high light, excessive amounts of iron or sodium acetate, either singly or in combination. Meanwhile, four isoforms of MnSOD were detected by a nondenaturing polyacrylamide gel electrophoresis approach. In addition, an iron-containing superoxide dismutase (FeSOD) was detected in the cells using the same technique. Differential expressions of SOD isoenzymes under different abiotic stressors were also investigated and discussed, at both mRNA and protein levels.

Fe-superoxide dismutase and 2-hydroxy-1,4-benzoquinone reductase preclude the auto-oxidation step in 4-aminophenol metabolism by Burkholderia sp. strain AK-5

Burkholderia sp. strain AK-5 converts 4-aminophenol to maleylacetic acid via 1,2,4-trihydroxybenzene, which is unstable in vitro and non-enzymatically auto-oxidized to 2-hydroxy-1,4-benzoquinone. Crude extract of strain AK-5 retarded the auto-oxidation and reduced the substrate analogue, 2,6-dimethoxy-1,4-benzoquinone, in the presence of NADH. The two enzymes responsible were purified to homogeneity. The deduced amino acid sequence of the enzyme that inhibited the auto-oxidation showed a high level of identity to sequences of iron-containing superoxide dismutases (Fe-SODs) and contained a conserved metal-ion-binding site; the purified enzyme showed superoxide dismutase activity and contained 1 mol of Fe per mol of enzyme, identifying it as Fe-SOD. Among three type SODs tested, Fe-SOD purified here inhibited the auto-oxidation most efficiently. The other purified enzyme showed a broad substrate specificity toward benzoquinones, including 2-hydroxy-1,4-benzoquinone, converting them to the corresponding 1,4-benzenediols; the enzyme was identified as 2-hydroxy-1,4-benzoquinone reductase. The deduced amino acid sequence did not show a high level of identity to that of benzoquinone reductases from bacteria and fungi that degrade chlorinated phenols or nitrophenols. The indirect role of Fe-SOD in 1,2,4-trihydroxybenzene metabolism is probably to scavenge and detoxify reactive species that promote the auto-oxidation of 1,2,4-trihydroxybenzene in vivo. The direct role of benzoquinone reductase would be to convert the auto-oxidation product back to 1,2,4-trihydroxybenzene. These two enzymes together with 1,2,4-trihydroxybenzene 1,2-dioxygenase convert 1,2,4-trihydroxybenzene to maleylacetic acid.

Structural Analysis of Peroxide-Soaked MnSOD Crystals Reveals Side-On Binding of Peroxide to Active-Site Manganese

The superoxide dismutase (SOD) enzymes are important antioxidant agents that protect cells from reactive oxygen species. The SOD family is responsible for catalyzing the disproportionation of superoxide radical to oxygen and hydrogen peroxide. Manganese- and iron-containing SOD exhibit product inhibition whereas Cu/ZnSOD does not. Here, we report the crystal structure of Escherichia coli MnSOD with hydrogen peroxide cryotrapped in the active site. Crystallographic refinement to 1.55 Å and close inspection revealed electron density for hydrogen peroxide in three of the four active sites in the asymmetric unit. The hydrogen peroxide molecules are in the position opposite His26 that is normally assumed by water in the trigonal bipyramidal resting state of the enzyme. Hydrogen peroxide is present in active sites B, C, and D and is side-on coordinated to the active-site manganese. In chains B and D, the peroxide is oriented in the plane formed by manganese and ligands Asp167 and His26. In chain C, the peroxide is bound, making a 70° angle to the plane. Comparison of the peroxide-bound active site with the hydroxide-bound octahedral form shows a shifting of residue Tyr34 towards the active site when peroxide is bound. Comparison with peroxide-soaked Cu/ZnSOD indicates end-on binding of peroxide when the SOD does not exhibit inhibition by peroxide and side-on binding of peroxide in the product-inhibited state of MnSOD.

Did Trypanosomatid Parasites Contain a Eukaryotic Alga–Derived Plastid in Their Evolutionary Past?

The Trypanosomatidae is closely related to euglenids that harbor plastids acquired from a green alga via secondary endosymbiosis. This discovery led to the idea that trypanosomatid parasites contained a green alga-derived plastid in their evolutionary past, an evolutionary scenario that was criticized based on the rarity of plant/plastid/cyanobacterium-like genes in the completely sequenced genomes of Trypanosoma and Leishmania species. Because it is difficult to identify such genes, however, their apparent rarity does not preclude a previous plastid endosymbiosis in the Trypanosomatidae. The genome of the plastid-less apicomplexan Cryptosporidium parvum preserves only a handful of plant/plastid/cyanobacterium-like genes, suggesting massive loss of plastid genes after elimination of its plastid. Additional support for such wholesale gene loss comes from fucoxanthin-containing dinoflagellates. Trypanosomatid nuclear genomes contain cyanobacterium-, green plant-, and haptophyte alga-derived genes, suggesting that they could have possessed a plastid in their evolutionary past; however, these genes also could represent examples of more typical horizontal gene transfer that did not accompany a plastid endosymbiosis. Thus, the presence of host cell genes that were adapted for use in the plastid would be much stronger evidence for a past plastid endosymbiosis in the Trypanosomatidae. Good examples of such genes are those encoding superoxide dismutases (SODs). Trypanosomatid parasites possess 4 iron-containing SODs, with 2 of them, SODA and SODC, targeted to the mitochondrion. In contrast with SODAs with classical single-domain mitochondrial targeting signals, SODCs carry bipartite pre-sequences composed of a signal peptide, followed by a transit peptide. Interestingly, these N-terminal extensions show striking similarities in length, hydropathy profiles, amino acid composition, and targeting properties to pre-sequences of proteins targeted to eukaryotic alga-derived plastids of euglenids and dinoflagellates. In turn, phylogenetic analyses indicate that SODCs originated from a mitochondrion-targeted SOD via gene duplication and were inherited vertically in the trypanosomatid lineage. These data represent a new kind of evidence for a past plastid endosymbiosis in the Trypanosomatidae, but the nature of this plastid remains unclear. It is usually assumed that the trypanosomatid plastid shared a common origin with that of euglenids, but Delta 4 desaturase phylogenies suggest that it could have originated via an independent, tertiary endosymbiosis involving a haptophyte alga. It is also possible that ancestors of the Trypanosomatidae initially possessed a primary plastid that later was replaced by a secondary or tertiary plastid.

Superoxide interaction with nickel and iron superoxide dismutases

Interaction of superoxide with the metal active sites of nickel and iron-containing superoxide dismutases is investigated with density functional theory. Outer-sphere mechanisms for both the reduction and oxidation of superoxide are proposed to be the common feature of these structurally unrelated SODs.

Expression, purification and molecular modeling of iron-containing superoxide dismutase from Acidithiobacillus ferrooxidans

The superoxide dismutase(SOD) from Acidithiobacillus ferrooxidans may play an important role in its tolerance to the extremely toxic and oxidative environment of bioleaching. This gene was cloned and then successfully expressed in Escherichia coli. The expressed protein was finally purified by one-step affinity chromatography to homogeneity and observed to be dimer according to SDS-PAGE and MALDI-TOF-MS. The metal content determination and optical spectra results of the recombinant protein confirmed that the protein was an iron-containing superoxide dismutase. Molecular modeling for the protein revealed that the iron atom was ligated by His26, His75, Asp158 and His162.

Agrobacterium tumefaciensiron superoxide dismutases have protective roles against singlet oxygen toxicity generated from illuminated Rose Bengal

Singlet oxygen is a highly reactive form of molecular oxygen that is harmful to biological systems. Here, the role of three iron-containing superoxide dismutase (sodB) genes is clearly shown in protecting Agrobacterium tumefaciens against singlet oxygen toxicity. A sodBI mutant was more sensitive to singlet oxygen than both wild-type bacteria and a double sodBII-sodBIII mutant strain. Moreover, a sodBI-sodBII double mutant had higher sensitivity to singlet oxygen than a single sodBI mutant, although the double mutant was comparable to a sodB null mutant. High-level expression of sodBI and sodBII fully complemented the singlet oxygen hypersensitivity phenotype of the sodB null mutant, while high-level expression of sodBIII encoding a periplasmic SOD only partially restored the phenotype. Taken together, our data suggest that SodBI and SodBII have novel protective roles against singlet oxygen toxicity through unknown mechanisms.

Small RNA ArrF Regulates the Expression of sodB and feSII Genes in Azotobacter vinelandii

Azotobacter vinelandii contains a prrF-like sequence in a noncoding region of the chromosome. Like the Pseudomonas aeruginosa PrrF small RNA-encoding genes, the expression of the sequence, herein named arrF (Azotobacter regulatory RNA involving Fe), was increased 100-fold in wild-type cells in response to iron depletion. The level of ArrF was also increased to the same degree in the iron-replete fur mutant, but down back to a wild-type level when this fur mutant was complemented with the wild-type fur gene. These results, with the location of arrF gene in a noncoding region, suggest that this gene encodes an iron-responsive small RNA whose expression is negatively regulated by the Fur-Fe(2+) complex. Disruption of this arrF gene upregulated the expression of iron-containing superoxide dismutase and FeSII protein, whereas fur mutation or iron depletion decreased the level of their transcript. A short region in the 5'-untranslated region of each transcript was predicted to be quite complementary to the core sequence of ArrF, assuming that ArrF represses the expression of the genes under Fur control by an antisense RNA mechanism. However, unlike the P. aeruginosa PrrF that has extensive targets in the tricarboxylic acid cycle and glyoxylate cycle, ArrF had little effect on those genes. The findings that there is a poor overlap between ArrF and PrrF targets and that the FeSII gene, which is present only in the chromosome of nitrogen-fixing bacterial species, is controlled by ArrF suggest that ArrF might have unique targets, some of which are involved in N(2) fixation.

Were class C iron-containing superoxide dismutases of trypanosomatid parasites initially imported into a complex plastid? A hypothesis based on analyses of their N-terminal targeting signals

Trypanosomatid parasites possess 2 distinct iron-containing superoxide dismutases (Fe-SODs) designated SODA and SODC, both of which are targeted to their mitochondria. In contrast to SODAs that carry typical mitochondrial transit peptides, SODCs have highly unusual mitochondrial targeting signals. Our analyses clearly show that these pre-sequences are bipartite possessing a signal peptide-like domain followed by a transit peptide-like domain. Consequently, they resemble N-terminal extensions of proteins targeted to multi-membrane plastids, suggesting that trypanosomatids once contained a eukaryotic alga-derived plastid. Further support for this hypothesis comes from striking similarities in length, hydropathy profile, and amino acid composition of SODC pre-sequences to those of Euglena and dinoflagellate plastid proteins. To account for these data, we propose that the Trypanosomatidae initially possessed a gene encoding a mitochondrial Fe-SOD with a classical mitochondrial transit peptide. Before or after plastid acquisition, a gene duplication event gave rise to SODA and SODC. In a subsequent evolutionary step a signal peptide was linked to SODC, enabling its import into the plastid. When the trypanosomatid plastid subsequently was lost, natural selection favoured adaptation of the SODC N-terminal signal as a mitochondrial transit peptide and re-targeting to the mitochondrion.