The pH‐Dependent Changes of the Enzymic Activity and Spectroscopic Properties of Iron‐Substituted Manganese Superoxide Dismutase

Abstract

Manganese-containing superoxide dismutases (Mn-SODs) and iron-containing superoxide dismutases (Fe-SODS) from aerobic bacteria often show high metal specificity for their enzymic activities by a standard assay system using xanthine-xanthine oxidase and cytochrome c. In this study, we have attempted to characterize the structural basis of the metal specificity of manganese-containing SOD (Mn-SOD) using Fe-substituted Mn-SOD prepared from apo-Mn-SOD from Serratia marcescens. The Fe3+ content of the Fe-substituted enzyme was 1.71 t 0.14 mol/mol dimer and the specific activity was 34.8 -C 4.8 units . mg protein-' . mol Fe3+-' . mol subunit-'. Fe-substituted Mn-SOD was found to react with the superoxide anion at pH 8.1 with a second-order rate constant of 6X lo6 M-' SK', which is approximately 1 % of that Iof native Mn-SOD at the same pH. However, the rate constant increased with decreasing pH to approximately 10% (5X107 MK1 s-') that of native Mn-SOD at pH 6.0 with a pK of 7.0, The visible absoIption spectrum and EPR spectrum of Fe-substituted Mn-SOD also showed pH-dependent changes with pK values of 6.6 and 7.2, respectively. Similarly, the affinity of the azide ion, an analog of the superoxide ion, for iron of Fe-substituted Mn-SOD increased with decreasing pH, with a pK value of 7.0 (e.g. Kd =0.1 mM at pH 6 . 2 and 0.9 mM at pH 8.2). The similarity of these pK values suggests that the activity, the spectral changes and the affinity of the azide ion for iron are derived from the same change in the metal environment. After comparison with the reported pK values (around 9) of similar pH-dependent changes in the spectra, the enzymic activity and the affinity of azide for iron of Fe-SOD from Escherichia coli, we proposed that the difference in the pK values of a hydroxide ion binding to iron between Fe-substituted Mn-SOD and Fe-SOD may cause the different pH dependencies of these changes in each SOD.Keywords. Superoxide dismutase; manganese; iron-substitution; metal-specificity ; Serratia murcescens.Three classes of superoxide dismutase (SOD) have been reported, based on their active-site metals ; copper-and zinc-containing superoxide dismutase (Cu, Zn-SOD), iron-containing SOD (Fe-SOD) and manganese-containing SOD (Mn-SOD [5] have been shown to have similar amino acid sequences, three-dimensional structures and trigonal pyramidal metal-site geometries. The iron and manganese atoms in these Fe-SODS and Mn-SODS are commonly ligated by three histidine residues and one aspartic acid residue. Previous reconstitution experiments, however, indicated that the metal requirements for enzymic activity of the Fe-SODS from R ovalis 161 and Photobacterium leiognathi [7] Correspondence 10 F. Yamdkura, Department of Chemistry, School of Medicine, Juntendo University, 1-1 Hiraga-gakuendai, Inba-gun, Chiba, Japan 270-1 6Fax: +81 476 98 1036. Abbreviations. SOD, superoxide dismutase; Cu, Zn-SOD, copper and zinc-containing superoxide dismutase ; Fe-SOD, iron-containing superoxide dismutase ; hh-SOD, manganese-containing superoxide dism...

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“…The metal‐free apo‐protein, apoSodA, was then incubated with different amounts of MnCl 2 for reconstitution. [ 35,58 ] The incorporation of manganese was monitored using fluorescence readings, as the intrinsic tryptophan fluorescence changes due to structural changes during reconstitution (Figure 5a). [ 59 ] The dissociation constant K d was calculated from the obtained data (Figure 5b).…”

“…Subsequently, two steps of dialysis were performed: first, 5 ml of protein sample was dialyzed against 1‐L Tris buffer (20 mM tris/HCl, pH 7.5) containing 10‐mM EDTA, second against 1 L of Tris buffer without EDTA. [ 35 ]…”

A Single Outer-Sphere Mutation Stabilizes apo-Mn Superoxide Dismutase by 35 °C and Disfavors Mn Binding

“…Four different types of superoxide dismutases (SODs) are distinguished on the basis of the identity of their redox-active metal ion cofactor: the manganese-specific SODs (MnSODs), the iron-specific SODs (FeSODs), the copper- and zinc-containing SODs (CuZnSODs), and the nickel-containing SODs (NiSODs) . Although encoded by different genes in Escherichia coli, FeSOD and MnSOD are believed to have evolved from a common ancestor because they display homologous structures and amino acid sequences. − Amino acid conservation is particularly strong in the active site, with all four ligands to the metal ion being identically conserved in all FeSODs and MnSODs described to date. , Thus, it is not surprising that MnSODs can be prepared with Fe bound instead of Mn − and vice versa . , In each case native-like coordination geometry is retained, − with the major difference being Fe-substituted MnSOD’s higher affinity for small anions including OH – , consistent with the higher Lewis acidity of Fe 3+ compared with Mn 3+ . ,,− While a few so-called “cambialistic” Fe/MnSODs have been identified that display activity under physiological conditions regardless of whether Fe or Mn is bound, , the canonical FeSODs and MnSODs require that their cognate metal ion be bound in order to perform with optimal enzymatic activity. ,, Thus, FeSODs and MnSODs provide a unique vantage point for understanding the crucial interface between the protein and metal ion, wherein the protein tunes the metal ion reactivity and the metal ion gives rise to the enzyme’s signature activity.…”