Structural analyses of various Cu2+, Zn2+-superoxide dismutases by differential scanning calorimetry and Raman spectroscopy

Lepock, James R.; Arnold, Lee D.; Torrie, B. H.; Andrews, B.; Kruuv, J.

doi:10.1016/0003-9861(85)90380-7

Cited by 66 publications

(47 citation statements)

References 29 publications

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“…This is consistent with the observation for bovine apo-SOD, that this protein denatures at a temperature 45 ~ lower than the holo-enzyme (23). In contrast to this behaviour of the metalloenzyme superoxide dismutase, another zinc containing metalloenzyme, human carbonic anhydrase, only revealed a slight decrease in structural stability upon metal removal, as seen from hydrogen-deuterium exchange studies (32), and zinc removal from alkaline phosphatase of E. coli caused a similar small effect on the hydrogen-tritium exchange rates (5).…”

Section: Discussionsupporting

confidence: 92%

“…Apparently the copper atoms, beyond their catalytical role, are equally important as the zinc atoms for the structural stability of yeast Cu_,,Zn2-SOD. Conformational changes subsequent to coordination of both copper and zinc, are also suggested by visible absorption and MCD spectra of yeast E~,Co~-SOD and Cu~,Co~-SOD (11), and by an increase in thermal stability of 30 ~ and 21 ~ following partial reconstitution of bovine apo-SOD with copper and zinc, respectively (23). These values are consistent with the metal induced, very extended conformational changes demonstrated by the present exchange experiments.…”

Section: Discussionsupporting

confidence: 90%

“…This protection of the peptide hydrogens in the molecule against contact with solvent, is also reflected in the high stability of Cu,,Zn_,-SOD toward the denaturing influence of e.g. heat, alkali and urea (2,23).…”

Section: Discussionmentioning

confidence: 99%

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Hydrogen-deuterium exchange in yeast Cu2,Zn2-superoxide dismutase and apo-superoxide dismutase

Dreyer

Ottesen

Hvidt

1986

Carlsberg Res. Commun.

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Keywords: Yeast Cu2,Zn2-superoxide dismutase, apo-superoxide dismutase, hydrogendeuterium exchange, metal-dependent structural transition, E X 2 -m e c h a n i s mThe role of copper and zinc for the structural properties of yeast Cu2,Zn2-SOD was studied by measuring the hydrogen-deuterium exchange in peptide groups of Cu:,Zn:-SOD, Cu:,E2-SOD, E:,Zn2-SOD and apo-SOD. Apo-SOD displays remarkably rapid exchange kinetics as compared to Cu2,Zn2-SOD, indicating a less stable structural organization after metal removal. At neutral and alkaline pD the exchange rates are in agreement with the EX2 mechanism of exchange; the exchange rates at low pD suggest a structural transition. In Cu2,Zn2-SOD this transition occurs at pD about 4.5, and in apo-SOD at pD about 6.2, 1.7 pD units higher. Binding of either copper or zinc to apo-SOD results in reductions of the exchange rate, which is further reduced after full reconstitufion to Cu_~,Zn.,-SOD, demonstrating that copper and zinc are equally important for the structural stability of the protein molecule. I N T R O D U C T I O NCu_,,Znz-SOD from yeast is a member of a highly conserved group of enzymes found in the cytosol of eucaryotic cells, believed to act as scavengers of toxic superoxide ions, O~, formed during oxidative metabolism. Yeast Cu2,Zn2-SOD has a molecular weight of 31,900, and it is composed of two identical subunits each containing a bimetallic Cu-Zn-center, which is essential for the catalytic mechanism (8,15,22). The X-ray crystal structure of bovine erythrocyte Cu2,Zn2-SOD at 2A resolution shows that each subunit consists of an eight stranded [3-bartel of Greek key topology, suggesting a very rigid structure. The copper is liganded to four histidines in a distorted square plane, and the zinc is liganded to three histidines and an aspartic acid in a tetrahedral geometry (28). 'H-NMR studies indicate close similarity in the metal binding sites of bovine erythrocyte and yeast Cu2,Zn2-SOD (8), although a metal bridging histidine in the bovine enzyme is apparently not liganded to zinc in the yeast enzyme (3).It is now well established that copper is essential for the catalysis of superoxide dismutation, during which copper is alternately oxidized and Abbreviations: Cu_,,E2-SOD = di-copper superoxide dismutase; E2,Zn2-SOD = di-zinc superoxide dismutase; SOD = superoxide dismutase.Springer-Verlag

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Section: Discussionsupporting

confidence: 92%

Section: Discussionsupporting

confidence: 90%

See 1 more Smart Citation

Hydrogen-deuterium exchange in yeast Cu2,Zn2-superoxide dismutase and apo-superoxide dismutase

Dreyer

Ottesen

Hvidt

1986

Carlsberg Res. Commun.

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“…This corresponds to a large (Ն5.2 kcal͞mol) reduction in dimer stability, indicating that a higher fraction of SOD1 is monomeric under aggregation-enhancing conditions than under near-physiological conditions. Because identical M m estimates were obtained for 10 M SOD1 at pH 3.5 with and without a 24-h incubation (Table 1) dimer dissociation is presumably reversible, and the irreversible (33,34) step in the aggregation lies downstream of dimer dissociation. The reversibility of dimer dissociation under aggregation-promoting conditions was directly demonstrated by SEC (Fig.…”

Section: Figmentioning

confidence: 53%

The rate and equilibrium constants for a multistep reaction sequence for the aggregation of superoxide dismutase in amyotrophic lateral sclerosis

Khare

Caplow

Dokholyan

2004

Proc. Natl. Acad. Sci. U.S.A.

141

158

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Mutation-induced aggregation of the dimeric enzyme Cu, Zn superoxide dismutase 1 (SOD1) has been implicated in the familial form of the disease amyotrophic lateral sclerosis, but the mechanism of aggregation is not known. Here, we show that in vitro SOD1 aggregation is a multistep reaction that minimally consists of dimer dissociation, metal loss from the monomers, and oligomerization of the apo-monomers:where Dholo, Mholo, Mapo, and A are the holo-dimer, holo-monomer, apo-monomer, and aggregate, respectively. Under aggregationpromoting conditions (pH 3.5), the rate and equilibrium constants corresponding to each step are: A myotrophic lateral sclerosis (ALS) involves selective motor neuron death in the brain and spinal cord (1-6), initiating a progressive paralysis in midlife. Point mutations in the cytoplasmic homodimeric enzyme Cu, Zn superoxide dismutase 1 [SOD1, molecular mass (M m ) Ϸ 32 kDa] were identified as the primary cause of Ϸ20% cases of the familial form of ALS (FALS) (7,8) in contrast with the sporadic form. Approximately 90 distinct FALS mutations are known (9). The toxic gain-of-function of the mutants is believed to be associated with either intracellular misfolding and aggregation or oxidative damage caused by mutant SOD1-catalyzed aberrant reactions, although the two scenarios may not be mutually exclusive (10, 11). The aggregation hypothesis is supported by the observations that in both mice and cell culture models death of motor neurons is preceded by formation of cytoplasmic aggregates containing mutant SOD1 (12-15), SOD1 knockout mice do not develop motor neuron disease (16), and even Cu-depleted SOD1 mutants cause the disease in mice (17). Although aggregation of SOD1 has also been found in some cases of sporadic ALS (18)(19)(20), the sporadic disease is believed to have a different molecular basis (1). FALS and SOD1-linked sporadic ALS may belong to the general class of protein conformational disorders in which perturbation of protein folding leads to a relatively higher population of misfolded or partially folded (21) protein molecules, which then aggregate into regular (22) structured fibrils. Toxicity may arise because of the saturation of the cellular chaperone machinery by misfolded or aggregated SOD1 molecules (23).ALS is an age-related disease occurring in midlife or later, and it is not surprising that the in vitro aggregation of SOD1 solutions under physiological conditions is slow. Therefore, it is common to study SOD1 aggregation by perturbing the environmental conditions, lowering the pH to 3.5 (24), adding mild denaturants such as trifluoroethanol, or using heat treatment (25), such that disease-like aggregates can be detected on experimental time scales. Aggregation-prone FALS mutations and͞or loss of metals also decrease SOD1 stability (26,27), indicating that exposure to nonphysiological conditions, such as low pH, mimics the effect of mutation by similarly lowering the barrier for SOD1 aggregation. Although the rate of aggregation can be enhanced in vitro, the molecula...

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“…2 is a highly stable homodimeric metalloenzyme (1)(2)(3)(4). Each subunit forms a Greek key ␤-barrel structure that binds one zinc and one copper ion (5,6); the zinc contributes to the structural stability of the active site, and the copper is involved in the redox dismutation of the toxic superoxide free radical (7).…”

Section: Sodmentioning

confidence: 99%

Calorimetric Analysis of Thermodynamic Stability and Aggregation for Apo and Holo Amyotrophic Lateral Sclerosis-associated Gly-93 Mutants of Superoxide Dismutase

Stathopulos

Rumfeldt

Karbassi

et al. 2006

Journal of Biological Chemistry

Self Cite

129

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Differential scanning calorimetry was used to measure changes in thermodynamic stability and aggregation for glycine 93 mutants of human copper, zinc-superoxide dismutase (SOD). Glycine 93 is a conserved residue at position i ؉ 3 of a tight turn and has been found to be a mutational hot spot in familial amyotrophic lateral sclerosis (fALS). The fALS-associated mutations, G93A, G93S, G93R, G93D, and G93V, were made in a pseudo wild-type background containing no free cysteines, which prevented the formation of aberrant disulfide bonds upon thermal unfolding, and enabled quantitative thermodynamic analysis of the effects of the mutations. Thermal unfolding was highly reversible for all the SODs in both the fully metallated (holo) and metal-free (apo) forms. The data for all the holo-SODs and for the apo-pseudo-wild-type SOD were well fit by a 2-state unfolding model for native dimer (N 2 ) to two unfolded monomers (2U), N 2 7 2U. The holo-and apo-forms of the mutants are significantly destabilized (by 1.5-3.5 kcal mol ؊1 monomer) relative to the corresponding forms of pseudo wild-type, with the relative stabilities being correlated with statistical preferences for amino acids in this structural context. Although van't Hoff (⌬H vH ) to calorimetric (⌬H cal ) enthalpy ratios are close to unity for all the holo-SODs and for apo-pseudo-wild-type, consistent with a 2-state transition, ⌬H vH is considerably larger than ⌬H cal for all the apo-mutants. This suggests that the mutations cause apo-SOD to have an increased propensity to misfold or aggregate, which may be linked to increased toxic mutant SOD aggregation in fALS. SOD2 is a highly stable homodimeric metalloenzyme (1-4). Each subunit forms a Greek key ␤-barrel structure that binds one zinc and one copper ion (5, 6); the zinc contributes to the structural stability of the active site, and the copper is involved in the redox dismutation of the toxic superoxide free radical (7). ALS is a common, rapidly progressive adult motor neuron disease for which there is very little in the way of treatment and no cure (8). Mutations in SOD were linked to fALS in 1993 (9); to date, over 110 SOD mutations have been associated with the disease. Although mutant SOD fALS represents only a few percent of all ALS cases, this is the major known cause of the disease (10, 11). Mutant SODs have a gain of toxic function, the molecular mechanism of which remains unresolved (8). Recent attention has focused on increased toxic misfolding of mutant SOD as a cause for fALS, analogous to other protein misfolding diseases (12-15). Increased misfolding has been correlated with decreased protein stability; hence, a better understanding of the thermodynamic effects of fALS mutations may lead to valuable insights into the disease process.Wild-type human SOD contains 4 cysteines per monomer; in the native protein two of these (Cys-57 and Cys-146) form a conserved intra-subunit disulfide bond, and the other two (Cys-6 and Cys-111) remain as free cysteines. Thermal unfolding of wild-type SOD is highly i...

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Structural analyses of various Cu2+, Zn2+-superoxide dismutases by differential scanning calorimetry and Raman spectroscopy

Cited by 66 publications

References 29 publications

Hydrogen-deuterium exchange in yeast Cu2,Zn2-superoxide dismutase and apo-superoxide dismutase

Hydrogen-deuterium exchange in yeast Cu2,Zn2-superoxide dismutase and apo-superoxide dismutase

The rate and equilibrium constants for a multistep reaction sequence for the aggregation of superoxide dismutase in amyotrophic lateral sclerosis

Calorimetric Analysis of Thermodynamic Stability and Aggregation for Apo and Holo Amyotrophic Lateral Sclerosis-associated Gly-93 Mutants of Superoxide Dismutase

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