Activation of CuZn superoxide dismutases from Caenorhabditis elegans does not require the copper chaperone CCS

Reactive oxygen species are produced as the direct result of aerobic metabolism and can cause damage to DNA, proteins, and lipids. A principal defense against reactive oxygen species involves the superoxide dismutases (SOD) that act to detoxify superoxide anions. Activation of CuZn-SODs in eukaryotic cells occurs post-translationally and is generally dependent on the copper chaperone for SOD1 (CCS), which inserts the catalytic copper cofactor and catalyzes the oxidation of a conserved disulfide bond that is essential for activity. In contrast to other eukaryotes, the nematode Caenorhabditis elegans does not contain an obvious CCS homologue, and we have found that the C. elegans intracellular CuZn-SODs (wSOD-1 and wSOD-5) are not dependent on CCS for activation when expressed in Saccharomyces cerevisiae. CCS-independent activation of CuZn-SODs is not unique to C. elegans; however, this is the first organism identified that appears to exclusively use this alternative pathway. As was found for mammalian SOD1, wSOD-1 exhibits a requirement for reduced glutathione in CCS-independent activation. Unexpectedly, wSOD-1 was inactive even in the presence of CCS when glutathione was depleted. Our investigation of the cysteine residues that form the disulfide bond in wSOD-1 suggests that the ability of wSODs to readily form this disulfide bond may be the key to obtaining high levels of activation through the CCS-independent pathway. Overall, these studies demonstrate that the CuZn-SODs of C. elegans have uniquely evolved to acquire copper without the copper chaperone and this may reflect the lifestyle of this organism.     

The effects of mitochondrial iron homeostasis on cofactor specificity of superoxide dismutase 2

Many metalloproteins have the capacity to bind diverse metals, but in living cells connect only with their cognate metal cofactor. In eukaryotes, this metal specificity can be achieved through metal-specific metallochaperone proteins. Herein, we describe a mechanism whereby Saccharomyces cerevisiae manganese superoxide dismutase (SOD2) preferentially binds manganese over iron based on the differential bioavailability of these ions within mitochondria. The bulk of mitochondrial iron is normally unavailable to SOD2, but when mitochondrial iron homeostasis is disrupted, for example, by mutations in S. cerevisiae mtm1, ssq1 and grx5, iron accumulates in a reactive form that potently competes with manganese for binding to SOD2, inactivating the enzyme. Studies in mtm1 mutants indicate that iron inactivation of SOD2 involves the Mrs3p/Mrs4p mitochondrial carriers and iron-binding frataxin (Yfh1p). A small pool of SOD2-reactive iron also exists under normal iron homeostasis conditions and binds SOD2 when mitochondrial manganese is low. The ability to control this reactive pool of iron is critical to maintaining SOD2 activity and has important potential implications for oxidative stress in disorders of iron overload.     

Superoxide Dismutase in Plants

Superoxide dismutases (SODs) are metal-containing enzymes that catalyze the dismutation of superoxide radicals to oxygen and hydrogen peroxide. The enzyme has been found in all aerobic organisms examined where it plays a major role in the defense against toxic-reduced oxygen species, which are generated as byproducts of many biological oxidations. The generation of oxygen radicals can be further exacerbated during environmental adversity and consequently SOD has been proposed to be important for plant stress tolerance. In plants, three forms of the enzyme exist, as classified by their active site metal ion: copper/zinc, manganese, and iron forms. The distribution of these enzymes has been studied both at the subcellular level and at the phylogenic level. It is only in plants that all three different types of SOD coexist. Their occurrence in the different subcellular compartments of plant cells allows a study of their molecular evolution and the possibility of understanding why three functionally equivalent but structurally different types of SOD have been maintained. Several cDNA sequences that encode the different SODs have recently become available, and the use of molecular techniques have greatly increased our knowledge about this enzyme system and about oxidative stress in plants in general, such that now is an appropriate time to review our current knowledge.     

The effects of glutaredoxin and copper activation pathways on the disulfide and stability of Cu,Zn superoxide dismutase

Mutations in Cu,Zn superoxide dismutase (SOD1) can cause amyotrophic lateral sclerosis (ALS) through mechanisms proposed to involve SOD1 misfolding, but the intracellular factors that modulate folding and stability of SOD1 are largely unknown. By using yeast and mammalian expression systems, we demonstrate here that SOD1 stability is governed by post-translational modification factors that target the SOD1 disulfide. Oxidation of the human SOD1 disulfide in vivo was found to involve both the copper chaperone for SOD1 (CCS) and the CCS-independent pathway for copper activation. When both copper pathways were blocked, wild type SOD1 stably accumulated in yeast cells with a reduced disulfide, whereas ALS SOD1 mutants A4V, G93A, and G37R were degraded. We describe here an unprecedented role for the thiol oxidoreductase glutaredoxin in reducing the SOD1 disulfide and destabilizing ALS mutants. Specifically, the major cytosolic glutaredoxin of yeast was seen to reduce the intramolecular disulfide of ALS SOD1 mutant A4V SOD1 in vivo and in vitro. By comparison, glutaredoxin was less reactive toward the disulfide of wild type SOD1. The apo-form of A4V SOD1 was highly reactive with glutaredoxin but not SOD1 containing both copper and zinc. Glutaredoxin therefore preferentially targets the immature form of ALS mutant SOD1 lacking metal co-factors. Overall, these studies implicate a critical balance between cellular reductants such as glutaredoxin and copper activation pathways in controlling the disulfide and stability of SOD1 in vivo.     

Copper modulates the degradation of copper chaperone for Cu,Zn superoxide dismutase by the 26 S proteosome

Copper chaperones are copper-binding proteins that directly insert copper into specific targets, preventing the accumulation of free copper ions that can be toxic to the cell. Despite considerable advances in the understanding of copper transfer from copper chaperones to their target, to date, there is no information regarding how the activity of these proteins is regulated in higher eukaryotes. The insertion of copper into the antioxidant enzyme Cu,Zn superoxide dismutase (SOD1) depends on the copper chaperone for SOD1 (CCS). We have recently reported that CCS protein is increased in tissues of rats fed copper-deficient diets suggesting that copper may regulate CCS expression. Here we show that whereas copper deficiency increased CCS protein in rats, mRNA level was unaffected. Rodent and human cell lines cultured in the presence of the specific copper chelator 2,3,2-tetraamine displayed a dose-dependent increase in CCS protein that could be reversed with the addition of copper but not iron or zinc to the cells. Switching cells from copper-deficient to copper-rich medium promoted the rapid degradation of CCS, which could be blocked by the proteosome inhibitors MG132 and lactacystin but not a cysteine protease inhibitor or inhibitors of the lysosomal degradation pathway. In addition, CCS degradation was slower in copper-deficient cells than in cells cultured in copper-rich medium. Together, these data show that copper regulates CCS expression by modulating its degradation by the 26 S proteosome and suggest a novel role for CCS in prioritizing the utilization of copper when it is scarce.     

A fraction of yeast Cu,Zn-superoxide dismutase and its metallochaperone, CCS, localize to the intermembrane space of mitochondria. A physiological role for SOD1 in guarding against mitochondrial oxidative damage

Cu,Zn-superoxide dismutase (SOD1) is an abundant, largely cytosolic enzyme that scavenges superoxide anions. The biological role of SOD1 is somewhat controversial because superoxide is thought to arise largely from the mitochondria where a second SOD (manganese SOD) already resides. Using bakers' yeast as a model, we demonstrate that Cu,Zn-SOD1 helps protect mitochondria from oxidative damage, as sod1Delta mutants show elevated protein carbonyls in this organelle. In accordance with this connection to mitochondria, a fraction of active SOD1 localizes within the intermembrane space (IMS) of mitochondria together with its copper chaperone, CCS. Neither CCS nor SOD1 contains typical N-terminal presequences for mitochondrial uptake; however, the mitochondrial accumulation of SOD1 is strongly influenced by CCS. When CCS synthesis is repressed, mitochondrial SOD1 is of low abundance, and conversely IMS SOD1 is very high when CCS is largely mitochondrial. The mitochondrial form of SOD1 is indeed protective against oxidative damage because yeast cells enriched for IMS SOD1 exhibit prolonged survival in the stationary phase, an established marker of mitochondrial oxidative stress. Cu,Zn-SOD1 in the mitochondria appears important for reactive oxygen physiology and may have critical implications for SOD1 mutations linked to the fatal neurodegenerative disorder, amyotrophic lateral sclerosis.     

Heterodimer formation between superoxide dismutase and its copper chaperone

Copper, zinc superoxide dismutase (SOD1) is activated in vivo by the copper chaperone for superoxide dismutase (CCS). The molecular mechanisms by which CCS recognizes and docks with SOD1 for metal ion insertion are not well understood. Two models for the oligomerization state during copper transfer have been proposed: a heterodimer comprising one monomer of CCS and one monomer of SOD1 and a dimer of dimers involving interactions between the two homodimers. We have investigated protein-protein complex formation between copper-loaded and apo yeast CCS (yCCS) and yeast SOD1 for both wild-type SOD1 (wtSOD1) and a mutant SOD1 in which copper ligand His 48 has been replaced with phenylalanine (H48F-SOD1). According to gel filtration chromatography, dynamic light scattering, analytical ultracentrifugation, and chemical cross-linking experiments, yCCS and this mutant SOD1 form a complex with the correct molecular mass for a heterodimer. No higher order oligomers were detected. Heterodimer formation is facilitated by the presence of zinc but does not depend on copper loading of yCCS. The complex formed with H48F-SOD1 is more stable than that formed with wtSOD1, suggesting that the latter is a more transient species. Notably, heterodimer formation between copper-loaded yCCS and wtSOD1 is accompanied by SOD1 activation only in the presence of zinc. These findings, taken together with structural, biochemical, and genetic studies, strongly suggest that in vivo copper loading of yeast SOD1 occurs via a heterodimeric intermediate.     

Multiple protein domains contribute to the action of the copper chaperone for superoxide dismutase.

The copper chaperone for superoxide dismutase (SOD1) inserts the catalytic metal cofactor into SOD1 by an unknown mechanism. We demonstrate here that this process involves the cooperation of three distinct regions of the copper chaperone for SOD1 (CCS): an amino-terminal Domain I homologous to the Atx1p metallochaperone, a central portion (Domain II) homologous to SOD1, and a short carboxyl-terminal peptide unique to CCS molecules (Domain III). These regions fold into distinct polypeptide domains as revealed through proteolysis protection studies. The biological roles of the yeast CCS domains were examined in yeast cells. Surprisingly, Domain I was found to be necessary only under conditions of strict copper limitation. Domain I and Atx1p were not interchangeable in vivo, underscoring the specificity of the corresponding metallochaperones. A putative copper site in Domain II was found to be irrelevant to yeast CCS activity, but SOD1 activation invariably required a CXC in Domain III that binds copper. Copper binding to purified yeast CCS induced allosteric conformational changes in Domain III and also enhanced homodimer formation of the polypeptide. Our results are consistent with a model whereby Domain I recruits cellular copper, Domain II facilitates target recognition, and Domain III, perhaps in concert with Domain I, mediates copper insertion into apo-SOD1.     

Heterodimeric structure of superoxide dismutase in complex with its metallochaperone

The copper chaperone for superoxide dismutase (CCS) activates the eukaryotic antioxidant enzyme copper, zinc superoxide dismutase (SOD1). The 2.9 A resolution structure of yeast SOD1 complexed with yeast CCS (yCCS) reveals that SOD1 interacts with its metallochaperone to form a complex comprising one monomer of each protein. The heterodimer interface is remarkably similar to the SOD1 and yCCS homodimer interfaces. Striking conformational rearrangements are observed in both the chaperone and target enzyme upon complex formation, and the functionally essential C-terminal domain of yCCS is well positioned to play a key role in the metal ion transfer mechanism. This domain is linked to SOD1 by an intermolecular disulfide bond that may facilitate or regulate copper delivery.     

The Right to Choose: Multiple Pathways for Activating Copper,Zinc Superoxide Dismutase

Since the discovery of SOD1 in 1969, there have been numerous achievements made in our understanding of the enzyme''s biochemical reactivity and its role in oxidative stress protection and as a genetic determinant in amyotrophic lateral sclerosis. Many recent advances have also been made in understanding the “activation” of SOD1, i.e. the process by which an inert polypeptide is converted to a mature active enzyme through post-translational modifications. To date, two such activation pathways have been identified: one requiring the CCS copper chaperone and one that works independently of CCS to insert copper and activate SOD1 through oxidation of an intramolecular disulfide. Depending on an organism''s lifestyle and complexity, different eukaryotes have evolved to favor one pathway over the other. Some organisms rely solely on CCS for activating SOD1, and others can only activate SOD1 independently of CCS, whereas the majority of eukaryotes appear to have evolved to use both pathways. In this minireview, we shall highlight recent advances made in understanding the mechanisms by which the CCS-dependent and CCS-independent pathways control the activity, structure, and intracellular localization of copper,zinc superoxide dismutase, with relevance to amyotrophic lateral sclerosis and an emphasis on evolutionary biology.     

The primary structure of superoxide dismutase purified from anaerobically maintained Bacteroides gingivalis

The superoxide dismutase (SOD) of Bacteroides gingivalis can use either iron or manganese as a cofactor in its catalytic activity. In this study, the complete amino acid sequence of this SOD purified from anaerobically maintained B. gingivalis cells was determined. The proteins consisted of 191 amino acid residues and had a molecular mass of 21,500. The sequence of B. gingivalis SOD showed 44-51% homology with those for iron-specific SODs (Fe-SODs) and 40-45% homology with manganese-specific SODs (Mn-SODs) from several bacteria. However, this sequence homology was considerably less than that seen among the Fe-SOD (65-74%) or Mn-SOD family (42-60%). This indicates that B. gingivalis SOD, which accepts either iron or manganese as metal cofactor, is a structural intermediate between the Fe-SOD and Mn-SOD families.     

Copper activation of superoxide dismutase 1 (SOD1) in vivo. Role for protein-protein interactions with the copper chaperone for SOD1

Insertion of copper into superoxide dismutase 1 (SOD1) in vivo requires the copper chaperone for SOD1 (CCS). CCS encompasses three protein domains: copper binding Domains I and III at the amino and carboxyl termini, and a central Domain II homologous to SOD1. Using a yeast interaction mating system, yeast CCS was seen to physically interact with SOD1, and this interaction required sequences at the predicted dimer interface of CCS Domain II. Interactions with SOD1 also required sequences of Domain III, but not Domain I. Mutations were introduced at the dimer interface of yeast SOD1, and the corresponding mutant failed to interact with CCS. When loaded with copper independent of CCS, this mutant SOD1 exhibited superoxide scavenging activity, but was normally inactive in vivo because CCS failed to recognize the enzyme. Activation of SOD1 by CCS was also examined using an in vivo assay for copper incorporation into SOD1. Yeast CCS was observed to insert copper into a pre-existing pool of apoSOD1 without the need for new SOD1 synthesis or for protein unfolding by the major SSA cytosolic heat shock proteins. Our data are consistent with a model in which prefolded dimers of apoSOD1 serve as substrate for the CCS copper chaperone.     

Manganese superoxide dismutase in Saccharomyces cerevisiae acquires its metal co-factor through a pathway involving the Nramp metal transporter, Smf2p

Eukaryotes express both copper/zinc (SOD1)- and manganese (SOD2)-requiring superoxide dismutase enzymes that guard against oxidative damage. Although SOD1 acquires its copper through a specific copper trafficking pathway, nothing is known regarding the intracellular manganese trafficking pathway for SOD2. We demonstrate here that in Saccharomyces cerevisiae cells delivery of manganese to SOD2 in the mitochondria requires the Nramp metal transporter, Smf2p. SOD2 activity is greatly diminished in smf2Delta mutants, even though the mature SOD2 polypeptide accumulates to normal levels in mitochondria. Treating smf2Delta cells with manganese supplements corrected the SOD2 defect, as did elevating intracellular manganese through mutations in PMR1. Hence, manganese appears to be inaccessible to mitochondrial SOD2 in smf2 mutants. Cells lacking SMF2 also exhibited defects in manganese-dependent steps in protein glycosylation and showed an overall decrease in steady-state levels of accumulated manganese. By comparison, mutations in the cell surface Nramp transporter, Smf1p, had very little impact on manganese accumulation and trafficking. Smf2p resides in intracellular vesicles and shows no evidence of plasma membrane localization, even in an end4 mutant blocked for endocytosis. We propose a model in which Smf2p-containing vesicles play a central role in manganese trafficking to the mitochondria and other cellular sites as well.     

Copper chaperone-dependent and -independent activation of three copper-zinc superoxide dismutase homologs localized in different cellular compartments in Arabidopsis

Superoxide dismutases (SODs) are important antioxidant enzymes that catalyze the disproportionation of superoxide anion to oxygen and hydrogen peroxide to guard cells against superoxide toxicity. The major pathway for activation of copper/zinc SOD (CSD) involves a copper chaperone for SOD (CCS) and an additional minor CCS-independent pathway reported in mammals. We characterized the CCS-dependent and -independent activation pathways for three CSDs localized in different cellular compartments in Arabidopsis (Arabidopsis thaliana). The main activation pathway for CSD1 in the cytoplasm involved a CCS-dependent and -independent pathway, which was similar to that for human CSD. Activation of CSD2 in chloroplasts depended totally on CCS, similar to yeast (Saccharomyces cerevisiae) CSD. Peroxisome-localized CSD3 via a CCS-independent pathway was similar to nematode (Caenorhabditis elegans) CSD in retaining activity in the absence of CCS. In Arabidopsis, glutathione played a role in CCS-independent activation, as was reported in humans, but an additional factor was required. These findings reveal a highly specific and sophisticated regulation of CSD activation pathways in planta relative to other known CCS-independent activation.     

A gain of superoxide dismutase (SOD) activity obtained with CCS, the copper metallochaperone for SOD1

The incorporation of copper ions into the cytosolic superoxide dismutase (SOD1) is accomplished in vivo by the action of the copper metallochaperone CCS (copper chaperone for SOD1). Mammalian CCS is comprised of three distinct protein domains, with a central region exhibiting remarkable homology (approximately 50% identity) to SOD1 itself. Conserved in CCS are all the SOD1 zinc binding ligands and three of four histidine copper binding ligands. In CCS the fourth histidine is replaced by an aspartate (Asp(200)). Despite this conservation of sequence between SOD1 and CCS, CCS exhibited no detectable SOD activity. Surprisingly, however, a single D200H mutation, targeting the fourth potential copper ligand in CCS, granted significant superoxide scavenging activity to this metallochaperone that was readily detected with CCS expressed in yeast. This mutation did not inhibit the metallochaperone capacity of CCS, and in fact, D200H CCS appears to represent a bifunctional SOD that can self-activate itself with copper. The aspartate at CCS position 200 is well conserved among mammalian CCS molecules, and we propose that this residue has evolved to preclude deleterious reactions involving copper bound to CCS.     

Manganese activation of superoxide dismutase 2 in the mitochondria of Saccharomyces cerevisiae

Manganese-dependent superoxide dismutase 2 (SOD2) in the mitochondria plays a key role in protection against oxidative stress. Here we probed the pathway by which SOD2 acquires its manganese catalytic cofactor. We found that a mitochondrial localization is essential. A cytosolic version of Saccharomyces cerevisiae Sod2p is largely apo for manganese and is only efficiently activated when cells accumulate toxic levels of manganese. Furthermore, Candida albicans naturally produces a cytosolic manganese SOD (Ca SOD3), yet when expressed in the cytosol of S. cerevisiae, a large fraction of Ca SOD3 also remained manganese-deficient. The cytosol of S. cerevisae cannot readily support activation of Mn-SOD molecules. By monitoring the kinetics for metalation of S. cerevisiae Sod2p in vivo, we found that prefolded Sod2p in the mitochondria cannot be activated by manganese. Manganese insertion is only possible with a newly synthesized polypeptide. Furthermore, Sod2p synthesis appears closely coupled to Sod2p import. By reversibly blocking mitochondrial import in vivo, we noted that newly synthesized Sod2p can enter mitochondria but not a Sod2p polypeptide that was allowed to accumulate in the cytosol. We propose a model in which the insertion of manganese into eukaryotic SOD2 molecules is driven by the protein unfolding process associated with mitochondrial import.     

Yeast frataxin mutants display decreased superoxide dismutase activity crucial to promote protein oxidative damage

Iron overload is involved in several pathological conditions, including Friedreich ataxia, a disease caused by decreased expression of the mitochondrial protein frataxin. In a previous study, we identified 14 proteins selectively oxidized in yeast cells lacking Yfh1, the yeast frataxin homolog. Most of these were magnesium-binding proteins. Decreased Mn-SOD activity, oxidative damage to CuZn-SOD, and increased levels of chelatable iron were also observed in this model. This study explores the relationship between low SOD activity, the presence of chelatable iron, and protein damage. We observed that addition of copper and manganese to the culture medium restored SOD activity and prevented both oxidative damage and inactivation of magnesium-binding proteins. This protection was compartment specific: recovery of mitochondrial enzymes required the addition of manganese, whereas cytosolic enzymes were recovered by adding copper. Copper treatment also decreased Δyfh1 sensitivity to menadione. Finally, a Δsod1 mutant showed high levels of chelatable iron and inactivation of magnesium-binding enzymes. These results suggest that reduced superoxide dismutase activity contributes to the toxic effects of iron overloading. This would also apply to pathologies involving iron accumulation.     

Cellular iron distribution in Bacillus anthracis

Although successful iron acquisition by pathogens within a host is a prerequisite for the establishment of infection, surprisingly little is known about the intracellular distribution of iron within bacterial pathogens. We have used a combination of anaerobic native liquid chromatography, inductively coupled plasma mass spectrometry, principal-component analysis, and peptide mass fingerprinting to investigate the cytosolic iron distribution in the pathogen Bacillus anthracis. Our studies identified three of the major iron pools as being associated with the electron transfer protein ferredoxin, the miniferritin Dps2, and the superoxide dismutase (SOD) enzymes SodA1 and SodA2. Although both SOD isozymes were predicted to utilize manganese cofactors, quantification of the metal ions associated with SodA1 and SodA2 in cell extracts established that SodA1 is associated with both manganese and iron, whereas SodA2 is bound exclusively to iron in vivo. These data were confirmed by in vitro assays using recombinant protein preparations, showing that SodA2 is active with an iron cofactor, while SodA1 is cambialistic, i.e., active with manganese or iron. Furthermore, we observe that B. anthracis cells exposed to superoxide stress increase their total iron content more than 2-fold over 60 min, while the manganese and zinc contents are unaffected. Notably, the acquired iron is not localized to the three identified cytosolic iron pools.