Key amino acid positions involved in activity, heat stability and covalent modification of rat mitochondrial manganese superoxide dismutase

Mitochondrial Mn-SOD is encoded by the nuclear genome as a precursor headed by a signal peptide spanning 24 amino acid residues; this leader peptide is then removed for the entry of the mature enzyme in mitochondria. The rat mitochondrial Mn-SOD (ratSOD2), sharing 93% amino acid identity with the human counterpart, is an appropriate model to study the molecular and functional properties of this key mitochondrial enzyme. This investigation regards the role of some crucial amino acid positions of ratSOD2 regulating catalysis, reactivity and thermal resistance of the enzyme. In particular, the role of three amino acid residues, namely Q143, Y34 and S82, has been investigated. The study was carried out through the heterologous production of the mature form of ratSOD2 and its mutants obtained by sitedirected mutagenesis on the corresponding gene. Six recombinant forms of the enzyme were produced, carrying the Q143 or H143 residue with or without the Y34F or S82A replacement. All proteins bound manganese and were organized as homotetramers. A six-fold reduction of the activity was observed in ratSOD2 forms containing the H143 variant; on the other hand, the Y34F and S82A substitutions only caused a modest reduction of enzymatic activity, compared to the Q143 form. Heat inactivation studies showed the high thermo-tolerance of ratSOD2 and allowed an evaluation of the related activation parameters of inactivation. Compared to the Q143 variant, the H143 counterpart was significantly less heat stable and displayed moderately lower enthalpic and entropic factors; the Y34F substitution caused a moderate reduction of heat stability, whereas the S82A replacement slightly improved the thermo-tolerance of the Q143 variant; both substitutions significantly increased the enthalpic and entropic factors of heat inactivation, the greatest effect being observed with the S82A substitution. All recombinant forms of ratSOD2 were glutathionylated in Escherichia coli, a feature pointing to the high reactivity of ratSOD2 towards glutathione. Moreover, the S82 position of the enzyme was found phosporylated in an in vitro system containing human mitochondrial protein extracts as source of protein kinases. These data highlight the role played by some critical residues of ratSOD2 and suggest fine levels of regulation occurring in vivo.