The depletion of superoxide catalyzed by human manganese superoxide dismutase (MnSOD) was observed spectrophotometrically by measuring the absorbance of superoxide at 250 -280 nm following pulse radiolysis and by stopped-flow spectrophotometry. Catalysis showed an initial burst of activity lasting approximately 1 ms followed by the rapid emergence of a greatly inhibited catalysis of zero-order rate. These catalytic properties of human MnSOD are qualitatively similar to those reported for MnSOD from Thermus thermophilus (Bull, C., Niederhoffer, E. C., Yoshida, T., and Fee, J. ) showed a strong dependence on pH that could be described by an ionization of pK a 9.4 ؎ 0.1 with a maximum at low pH.Mitochondria are responsible for an extensive consumption of oxygen in cells, and the mitochondrial respiratory chain subsequently causes a large flux of oxygen radicals, a potential source of damage to protein and especially DNA. It is the mitochondrial superoxide dismutase (SOD) 1 that appears to be responsible for protection of these organelles from oxidative damage by catalyzing the decay of the superoxide radical anion.Three forms of SOD have different catalytic metal ions and different distributions (Beyer et al., 1991;Tainer et al., 1991); it is the manganese SOD that is found in mitochondria and prokaryotes, FeSOD is found in prokaryotes, and Cu,ZnSOD occurs primarily in eukaryotes but has been found in some bacteria. The crystal structure of human MnSOD has been determined at 2.2 Å resolution (Borgstahl et al., 1992). It shows the active sites grouped in pairs across the dimer interfaces, imposing a trigonal bipyramidal geometry on the manganese (Borgstahl et al., 1992). The crystal structures of the MnSOD from Thermus thermophilus at 1.8 Å (Ludwig et al., 1991) and from Bacillus stearothermophilus at 2.4 Å (Parker and Blake, 1988) show strong structural similarity with the human enzyme with a similar geometry about the metal. These structures are also similar to that of the FeSOD (Stoddard et al., 1990) including the same ligands to and geometry about the metal.It is now widely accepted that superoxide dismutases carry out catalysis through a redox process in which the metal cycles between oxidized and reduced states.Initial studies using pulse radiolysis of the MnSOD from B. stearothermophilus determined that its catalysis was complicated by the presence of an inactive form of the enzyme that can interconvert to an active form (McAdam et al., 1977a(McAdam et al., , 1977b. Steady-state constants for catalysis by MnSOD from T. thermophilus were obtained by Bull et al. (1991) from stoppedflow experiments, and the rapid emergence of the inactive form of the enzyme was quantitated. Bull et al. (1991) were able to observe the inactive form spectrophotometrically and suggested that it results from oxidative addition of O 2 . to the Mn(II) form of the enzyme, resulting in a side-on complex of Mn(III) and peroxide, Mn(III)O 2 Ϫ2 . We have overexpressed human MnSOD in Escherichia coli and purified this enzyme to homogenei...

The mitochondrial enzyme, manganese superoxide dismutase (MnSOD) is an integral component of the cell's defense against superoxide-mediated cellular damage. We have isolated and characterized four cDNA clones and the structural gene for rat MnSOD. Northern analyses using MnSOD cDNA probes detected at least five mRNAs in all tissues and cell types examined. Southern and Northern analysis using a 3' non-coding sequence probe, common to all the cDNAs, showed hybridization only to genomic restriction fragments that correspond to our genomic clone and the five MnSOD mRNAs. These data demonstrate that all of the rat MnSOD transcripts are derived from a single functional gene. Primer extension data indicate that transcription initiation is clustered within a few bases. Northern analysis using intron probes demonstrates that all five transcripts are fully processed. Northern analysis using cDNA and genomic probes from sequences progressively 3' to the end of the coding sequence indicates that size heterogeneity in the MnSOD transcripts results from variations in the length of the 3' non-coding sequence. From this data and the location of potential polyadenylation signals near the expected sites of transcript termination, we conclude that the existence of multiple MnSOD mRNA species originate as the result of alternate polyadenylation.

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