In vitro preparation of iron-substituted human manganese superoxide dismutase: Possible toxic properties for mitochondria

We prepared an iron-substituted form of recombinant human manganese superoxide dismutase (MnSOD) by using guanidine hydrochloride for the first time as a model of iron-misincorporated MnSOD, the formation of which has been reported by M. Yang et al. upon disruption of mitochondrial metal homeostasis in yeast (Yang et al. 2006, EMBO J. 25, 1775–1783). The iron-substituted enzyme contained 0.79 g atoms of Fe/mol of subunits and had a specific activity of 80 units/mg protein/g atom of Fe/mol of subunit, which was less than 3% of the activity of the purified MnSOD. Fe-substituted MnSOD (Fe-MnSOD) showed the same absorption spectrum as that of bacterial Fe-MnSODs reported, a similar pH-dependent change of the enzymatic activity, and a similar electron paramagnetic resonance spectrum. Fe-MnSOD showed more thermal stability than native MnSOD. The Fe-substituted enzyme showed a hydrogen-peroxide-mediated radical-generating activity, which was monitored by a cation radical of 2,2′-azinobis-(3-ethylbenzthiazoline-6-sulfonate) formation similar to that of Cu,ZnSOD, but native human MnSOD and FeSOD showed no radical-generation ability. This evidence suggests that a substitution of Mn to Fe in human MnSOD in mitochondria may produce a disadvantage for oxidative stress in three ways: loss of the enzymatic activity, increase of stability, and gain of radical-generating ability.