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Review
. 2006 Jul;1763(7):747-58.
doi: 10.1016/j.bbamcr.2006.05.003. Epub 2006 May 17.

Activation of superoxide dismutases: putting the metal to the pedal

Valeria Cizewski Culotta  1 , Mei Yang, Thomas V O'Halloran
Affiliations
Review

Activation of superoxide dismutases: putting the metal to the pedal

Valeria Cizewski Culotta et al. Biochim Biophys Acta. 2006 Jul.

Abstract

Superoxide dismutases (SOD) are important anti-oxidant enzymes that guard against superoxide toxicity. Various SOD enzymes have been characterized that employ either a copper, manganese, iron or nickel co-factor to carry out the disproportionation of superoxide. This review focuses on the copper and manganese forms, with particular emphasis on how the metal is inserted in vivo into the active site of SOD. Copper and manganese SODs diverge greatly in sequence and also in the metal insertion process. The intracellular copper SODs of eukaryotes (SOD1) can obtain copper post-translationally, by way of interactions with the CCS copper chaperone. CCS also oxidizes an intrasubunit disulfide in SOD1. Adventitious oxidation of the disulfide can lead to gross misfolding of immature forms of SOD1, particularly with SOD1 mutants linked to amyotrophic lateral sclerosis. In the case of mitochondrial MnSOD of eukaryotes (SOD2), metal insertion cannot occur post-translationally, but requires new synthesis and mitochondrial import of the SOD2 polypeptide. SOD2 can also bind iron in vivo, but is inactive with iron. Such metal ion mis-incorporation with SOD2 can become prevalent upon disruption of mitochondrial metal homeostasis. Accurate and regulated metallation of copper and manganese SOD molecules is vital to cell survival in an oxygenated environment.

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Figures

Fig. 1
Fig. 1
Proposed mechanism of copper insertion into SOD1 by its metallochaperone, CCS. The copper chaperone acquires copper through unknown routes and then docks with a disulfide reduced form of SOD1 (steps I and II). This complex is inert to further reaction unless exposed to oxygen or superoxide (step III), at which point a disulfide-linked heterodimeric intermediate forms. An analogous complex containing mutant SOD1 has been trapped and structurally characterized. In the case of the WT protein, this complex undergoes disulfide isomerization to an intramolecular disulfide in SOD1 (step IV). Copper is transferred at some point after introduction of oxygen and the mature monomer is proposed to be released from CCS. The left side of the image depicts several immature states of the protein in which the essential disulfide bond has not yet formed. If the conserved Cys are oxidized to form incorrect disulfide linkages, this can lead to SOD crosslinking and aggregation.
Fig. 2
Fig. 2
Oxidative aggregation model for ALS-linked mutations in SOD1: competition between folding and disulfide crosslinking pathways. The lower pathway traces the maturation of the WT SOD1, while the upper pathway depicts reactions of ALS-causing mutant SOD1. The disulfide-reduced apo-SOD1 molecules emerge from the ribosome and undergo folding and modification processes. In the case of the WT protein, the polypeptide folds and once zinc is acquired equilibrates between monomer and dimer forms. Both zinc binding and intra-molecular disulfide formation lend significant additional thermodynamic stability to the dimer (see green box), however the enzyme is not active in the absence of copper. In contrast to the WT protein, the apo and reduced states of many ALS mutant SODs are unfolded or misfolded at physiological temperature. The mutants studied to date are also predisposed to oligomerization when they are in the E,E-hSOD1SH state. In the presence of oxidants, these oligomeric forms of SOD1 are susceptible to formation of disulfide-linked multimers. In this model, these oxidized multimers ultimately nucleate the formation of larger insoluble aggregates that may contain folded or more mature forms of the protein such as those shown in the blue box. Formation of these oxidatively crosslinked aggregates are proposed to cause neuronal cell death. Under normal conditions the cellular machinery can degrade aggregates stabilized by non-covalent interactions. If, however, these oligomers experience even mild oxidative stress, they under go covalent crosslink formation which stabilizes the aggregates and leads to precipitation. Once the rate of insoluble aggregate formation outpaces the rate of degradation by the cellular quality control machinery, the cell or compartment may incur damage. Thus, any stress events that increase the expression of the most immature forms of the mutant SOD1 protein (see first branchpoint above) or otherwise increases its concentration (for instance by reverse of the lower path) may stimulate the rate of formation of robust aggregates. This model for SOD1-linked ALS thus involves two key steps: (a) oligomerization of misfolded reduced forms of the protein, (b) oxidation of the oligomers to form covalently linked insoluble aggregates that initiate cell death. Recent observations provide evidence for oxidative crosslinked aggregates in mitochondria of afflicted spinal cord neurons.
Fig. 3
Fig. 3
Role for mitochondrial import in metallation of SOD2. Shown is a model by which the translation, mitochondrial import and metal insertion of SOD2 are coupled. As the polypeptide enters mitochondria, SOD2 remains sufficiently unfolded to allow manganese insertion. Following manganese binding and complete entry into the mitochondrial matrix, the polypeptide can assemble into the quaternary enzyme. The mitochondrial carrier transporter Mtm1p helps prevent iron from interacting with SOD2. The substrate for transport by Mtm1p is unknown. Mitochondrial manganese is derived from intracellular vesicles harboring the Nramp manganese transporter, Smf2p.
Fig. 4
Fig. 4
Metal binding to SOD2 is determined by the differential bioavailability of iron versus manganese. Shown is a model for how changes in mitochondrial iron and manganese homeostasis can change metal specificity in SOD2. Under normal conditions, mitochondrial iron is in vast excess over total mitochondrial manganese (by 1–2 orders of magnitude), but this iron largely exists in a “SOD2-inert” state (green boxes) that is unavailable to SOD2. The small fraction of “SOD2-reactive” iron (red circles) cannot compete well with manganese (violet circles) for binding to SOD2. However, with disruptions in iron homeostasis caused by mtm1 mutations or by specific defects in Fe–S cluster assembly, SOD2-reactive iron levels substantially rise, and this iron competes well with manganese for binding to SOD2. Under manganese deficiency conditions, the small level of SOD2-reactive iron gains access to the active site of SOD2.
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