Zinc-coordination of aspartic acid-76 in Sulfolobus ferredoxin is not required for thermal stability of the molecule.
The highly thermostable 7Fe-ferredoxin from Sulfolobus sp. strain 7 has tightly bound zinc at the interface between the N-terminal extra domain and the C-terminal core. The zinc is tetrahedrally ligated by His-16, His-19, His-34, and Asp-76. Previous studies on truncated mutants have shown that the zinc and certain parts, i.e. not all, of the N-terminal extra stretch are responsible for the thermal stabilization of the molecule. To study the role of Asp-76, a series of mutants were constructed with Asp-76 replaced by Glu (D76E), Asn (D76N), or Ala (D76A). All the mutants, as well as wild type ferredoxin, bound 1 mol zinc/mol protein, and showed similar kinetics for 2-oxoacid:ferredoxin oxidoreductase. The stability of the protein was examined by thermal degradation of the clusters. In the absence of guanidium thiocyanate, the T(m), defined as the mid-point temperature of the thermal transition from the native to the denatured state, for every mutant was above 100 degrees C. The T(m) values in the presence of 1 M guanidium thiocyanate were determined to be 90.8, 90.2, 87.1, 84.4, and 72.9 degrees C for the natural, recombinant, D76N-, D76A-, and D76E-ferredoxins, respectively. These results indicate that the interaction between zinc and the carboxyl oxygen of Asp-76 has subtle effects on both the zinc-ligation and stability, although the native zinc center is liganded with high symmetry, suggesting that the three His residues are more important for zinc-binding.
Kojoh
Kanehisa
K
Department of Biotechnology, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, 113-8657, Tokyo, Japan.
Fukuda
Eriko
E
Matsuzawa
Hiroshi
H
Wakagi
Takayoshi
T
D001224:Aspartic Acid / Q000378:metabolism*; D005288:Ferredoxins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000235:genetics; D011487:Protein Conformation; D011489:Protein Denaturation; D013329:Structure-Activity Relationship; D016944:Sulfolobus / Q000737:chemistry*; D013816:Thermodynamics; D015032:Zinc / Q000378:metabolism*
D005288:Ferredoxins; D001224:Aspartic Acid; D015032:Zinc