Murine extracellular superoxide dismutase is converted into the inactive fold by the Ser195Cys mutation

Biochemistry. 2013 May 14;52(19):3369-75. doi: 10.1021/bi400171b. Epub 2013 May 1.


We have previously shown that human extracellular superoxide dismutase (EC-SOD) exists as two variants with differences in their disulfide bridge patterns: one form is the active enzyme (aEC-SOD), and the other is inactive (iEC-SOD). The availability of both active and inactive folding variants significantly reduces the specific activity of EC-SOD in vivo. Both forms are produced during biosynthesis, but the underlying folding mechanisms remain unclear. To address this issue, we expressed EC-SOD in heterologous systems that do not endogenously express iEC-SOD. Rodents express only aEC-SOD because they lack Cys195 (human EC-SOD sequence numbering), which is essential for the formation of iEC-SOD. However, cultured hamster cells and transgenic mice expressing human EC-SOD were able to produce both human a- and iEC-SOD variants, which led us to hypothesize that the folding was sequence-dependent rather than a property of the expression system. To substantiate this hypothesis, we expressed murine EC-SOD in a human cell line, and as expected, only aEC-SOD was produced. Significantly, when Cys195 was introduced, both murine aEC-SOD and a novel murine iEC-SOD were generated, and the specific activity of the murine EC-SOD was significantly reduced by the mutation. Collectively, these data suggest that Cys195 actuates the formation of iEC-SOD, independent of the expression system or host. In addition, the dual-folding pathway most likely requires biosynthesis factors that are common to both humans and rodents.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Substitution
  • Animals
  • CHO Cells
  • Cricetinae
  • Cricetulus
  • Cysteine / chemistry
  • HEK293 Cells
  • Humans
  • Kinetics
  • Mice
  • Mice, Transgenic
  • Mutagenesis, Site-Directed
  • Protein Folding
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism
  • Spectrometry, Mass, Electrospray Ionization
  • Superoxide Dismutase / chemistry*
  • Superoxide Dismutase / genetics*
  • Superoxide Dismutase / metabolism
  • Tandem Mass Spectrometry


  • Recombinant Proteins
  • SOD3 protein, human
  • Sod3 protein, mouse
  • Superoxide Dismutase
  • Cysteine