The hyper-thermostable Fe-superoxide dismutase from the archaeon Acidianusambivalens: Characterization, recombinant expression, crystallization and effects of metal exchange

An iron-containing superoxide dismutase (SOD; EC 1.15.1.1) of the hyperther mophilic archaeon Acidianus ambivalens (Aa-SOD) has been purified and chara cterized and the gene has been cloned and sequenced. The SOD from the facul tatively aerobic member of the crenarchaeota could be expressed in E. coli. Both, the native as well as the heterologously over-produced protein turned out to have extraordinarily high melting temperatures of 128 degreesC and 124.5 degreesC, respectively. To the best of our knowledge, this is the hig hest directly measured melting temperature of a native protein. Surprisingl y, neither the native nor the recombinant superoxide dismutase displays 100 % occupation of the metal coordination sites. Obviously it is not the incor poration of a metal ion that confers the extreme thermostability. Expressio n of the superoxide dismutase in the presence of different metals such as F e, Co, Ni, Mn and Cu offered the possibility of studying the hitherto unkno wn cofactor preference of iron-superoxide dismutase. The recombinant enzyme displayed the highest preference for incorporation of cobalt although iron is used as the natural cofactor. Spectroscopic analysis by EPR, atomic abs orption and UV/Vis spectroscopy as well as activity measurements and differ ential scanning calorimetry of the metal substituted superoxide dismutases were performed. However, the superoxide dismutase of A. ambivalens is activ e only with iron but may incorporate other metals equally well in the catal ytic center without loss of conformational stability or heat tolerance. The co-form of the enzyme could be crystallized.