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.
Yamakura
Fumiyuki
F
Department of Chemistry, Juntendo University School of Medicine, Inba, Chiba 270-1606, Japan. yamakura@sakura.juntendo.ac.jp
Kobayashi
Kazuo
K
Furukawa
Satoshi
S
Suzuki
Yasunori
Y
D052160:Benzothiazoles / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy; D004795:Enzyme Stability; D006358:Hot Temperature; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007501:Iron / Q000633:toxicity*; D008928:Mitochondria / Q000187:drug effects*; D013057:Spectrum Analysis; D013451:Sulfonic Acids / Q000378:metabolism; D013482:Superoxide Dismutase / Q000737:chemistry* / Q000302:isolation & purification / Q000633:toxicity
D052160:Benzothiazoles; D013451:Sulfonic Acids; C002502:2,2'-azino-di-(3-ethylbenzothiazoline)-6-sulfonic acid; D007501:Iron; D013482:Superoxide Dismutase