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Tetramerization reinforces the dimer interface of MnSOD.

Sheng Y, Durazo A, Schumacher M, Gralla EB, Cascio D, Cabelli DE, Valentine JS - PLoS ONE (2013)

Bottom Line: Although CaMnSODc was found to crystallize as a tetramer, there is no indication from the solution properties that the functionality of CaMnSODc in vivo depends upon the formation of the tetrameric structure.Dimeric CaMnSODc was found to be significantly more subject to thermal or denaturant-induced unfolding than tetrameric ScMnSOD.We conclude that the tetrameric assembly strongly reinforces the dimer interface, which is critical for MnSOD activity.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, University of California Los Angeles, Los Angeles, California, United States of America.

ABSTRACT
Two yeast manganese superoxide dismutases (MnSOD), one from Saccharomyces cerevisiae mitochondria (ScMnSOD) and the other from Candida albicans cytosol (CaMnSODc), have most biochemical and biophysical properties in common, yet ScMnSOD is a tetramer and CaMnSODc is a dimer or "loose tetramer" in solution. Although CaMnSODc was found to crystallize as a tetramer, there is no indication from the solution properties that the functionality of CaMnSODc in vivo depends upon the formation of the tetrameric structure. To elucidate further the functional significance of MnSOD quaternary structure, wild-type and mutant forms of ScMnSOD (K182R, A183P mutant) and CaMnSODc (K184R, L185P mutant) with the substitutions at dimer interfaces were analyzed with respect to their oligomeric states and resistance to pH, heat, and denaturant. Dimeric CaMnSODc was found to be significantly more subject to thermal or denaturant-induced unfolding than tetrameric ScMnSOD. The residue substitutions at dimer interfaces caused dimeric CaMnSODc but not tetrameric ScMnSOD to dissociate into monomers. We conclude that the tetrameric assembly strongly reinforces the dimer interface, which is critical for MnSOD activity.

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RP-mutant CaMnSODc is more subject to inactivation by pH than the wild type.Rate constants as a function of pH were determined by fitting the disappearances of low doses of O2− ([O2−]:[MnSOD] from 1–3) to first-order processes. The enzymes were WT ScMnSOD (solid triangle), K182R, A183P ScMnSOD (hollow triangle), WT CaMnSODc (solid circle) and K184R, L185P CaMnSODc (hollow circle). The data points circled and/or indicated with an arrow were measured after the pH was adjusted from 9–9.5 to neutral. The sample solutions contained 1 µM (in Mn) MnSOD in 10 mM potassium phosphate (pH 7), 10 mM sodium formate and 10 µM EDTA.
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pone-0062446-g004: RP-mutant CaMnSODc is more subject to inactivation by pH than the wild type.Rate constants as a function of pH were determined by fitting the disappearances of low doses of O2− ([O2−]:[MnSOD] from 1–3) to first-order processes. The enzymes were WT ScMnSOD (solid triangle), K182R, A183P ScMnSOD (hollow triangle), WT CaMnSODc (solid circle) and K184R, L185P CaMnSODc (hollow circle). The data points circled and/or indicated with an arrow were measured after the pH was adjusted from 9–9.5 to neutral. The sample solutions contained 1 µM (in Mn) MnSOD in 10 mM potassium phosphate (pH 7), 10 mM sodium formate and 10 µM EDTA.

Mentions: MnSOD catalytic activity (reactions 1 and 2) was measured when [O2−]:MnSOD ratio ranged from 1–3 to exclude any effect of product inhibition. We previously showed that inactivation occurs at a significantly lower pH in yeast MnSODs than in human MnSOD, with pKs (the pH at which the SOD activity drops by 50%) of 8.5 and 10.5, respectively [9]. Here, although both yeast MnSODs were engineered to imitate human MnSOD, neither of the mutant proteins gained stability at higher pH compared to the WT enzymes (Figure 4). The profile of RP-mutant ScMnSOD activity as a function of pH closely resembles that of WT ScMnSOD (Figure 4). The same mutations on CaMnSODc, however, resulted in an enzyme even more sensitive to pH, with the pK decreasing from ∼8.5 in the wild type to ∼8 in the mutant protein (Figure 4). Loss of activity at high pH was found to be reversible in WT yeast enzymes as well as in RP-mutant ScMnSOD, with a restoration of ∼50% of their original activity (Figure 4). By contrast, when the pH of the sample solution was adjusted from basic (≥9) to neutral, no restoration of activity was observed for RP-mutant CaMnSODc (Figure 4).


Tetramerization reinforces the dimer interface of MnSOD.

Sheng Y, Durazo A, Schumacher M, Gralla EB, Cascio D, Cabelli DE, Valentine JS - PLoS ONE (2013)

RP-mutant CaMnSODc is more subject to inactivation by pH than the wild type.Rate constants as a function of pH were determined by fitting the disappearances of low doses of O2− ([O2−]:[MnSOD] from 1–3) to first-order processes. The enzymes were WT ScMnSOD (solid triangle), K182R, A183P ScMnSOD (hollow triangle), WT CaMnSODc (solid circle) and K184R, L185P CaMnSODc (hollow circle). The data points circled and/or indicated with an arrow were measured after the pH was adjusted from 9–9.5 to neutral. The sample solutions contained 1 µM (in Mn) MnSOD in 10 mM potassium phosphate (pH 7), 10 mM sodium formate and 10 µM EDTA.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3646814&req=5

pone-0062446-g004: RP-mutant CaMnSODc is more subject to inactivation by pH than the wild type.Rate constants as a function of pH were determined by fitting the disappearances of low doses of O2− ([O2−]:[MnSOD] from 1–3) to first-order processes. The enzymes were WT ScMnSOD (solid triangle), K182R, A183P ScMnSOD (hollow triangle), WT CaMnSODc (solid circle) and K184R, L185P CaMnSODc (hollow circle). The data points circled and/or indicated with an arrow were measured after the pH was adjusted from 9–9.5 to neutral. The sample solutions contained 1 µM (in Mn) MnSOD in 10 mM potassium phosphate (pH 7), 10 mM sodium formate and 10 µM EDTA.
Mentions: MnSOD catalytic activity (reactions 1 and 2) was measured when [O2−]:MnSOD ratio ranged from 1–3 to exclude any effect of product inhibition. We previously showed that inactivation occurs at a significantly lower pH in yeast MnSODs than in human MnSOD, with pKs (the pH at which the SOD activity drops by 50%) of 8.5 and 10.5, respectively [9]. Here, although both yeast MnSODs were engineered to imitate human MnSOD, neither of the mutant proteins gained stability at higher pH compared to the WT enzymes (Figure 4). The profile of RP-mutant ScMnSOD activity as a function of pH closely resembles that of WT ScMnSOD (Figure 4). The same mutations on CaMnSODc, however, resulted in an enzyme even more sensitive to pH, with the pK decreasing from ∼8.5 in the wild type to ∼8 in the mutant protein (Figure 4). Loss of activity at high pH was found to be reversible in WT yeast enzymes as well as in RP-mutant ScMnSOD, with a restoration of ∼50% of their original activity (Figure 4). By contrast, when the pH of the sample solution was adjusted from basic (≥9) to neutral, no restoration of activity was observed for RP-mutant CaMnSODc (Figure 4).

Bottom Line: Although CaMnSODc was found to crystallize as a tetramer, there is no indication from the solution properties that the functionality of CaMnSODc in vivo depends upon the formation of the tetrameric structure.Dimeric CaMnSODc was found to be significantly more subject to thermal or denaturant-induced unfolding than tetrameric ScMnSOD.We conclude that the tetrameric assembly strongly reinforces the dimer interface, which is critical for MnSOD activity.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, University of California Los Angeles, Los Angeles, California, United States of America.

ABSTRACT
Two yeast manganese superoxide dismutases (MnSOD), one from Saccharomyces cerevisiae mitochondria (ScMnSOD) and the other from Candida albicans cytosol (CaMnSODc), have most biochemical and biophysical properties in common, yet ScMnSOD is a tetramer and CaMnSODc is a dimer or "loose tetramer" in solution. Although CaMnSODc was found to crystallize as a tetramer, there is no indication from the solution properties that the functionality of CaMnSODc in vivo depends upon the formation of the tetrameric structure. To elucidate further the functional significance of MnSOD quaternary structure, wild-type and mutant forms of ScMnSOD (K182R, A183P mutant) and CaMnSODc (K184R, L185P mutant) with the substitutions at dimer interfaces were analyzed with respect to their oligomeric states and resistance to pH, heat, and denaturant. Dimeric CaMnSODc was found to be significantly more subject to thermal or denaturant-induced unfolding than tetrameric ScMnSOD. The residue substitutions at dimer interfaces caused dimeric CaMnSODc but not tetrameric ScMnSOD to dissociate into monomers. We conclude that the tetrameric assembly strongly reinforces the dimer interface, which is critical for MnSOD activity.

Show MeSH
Related in: MedlinePlus