Zinc Binding Domain Stabilizes the Ferredoxin Molecule from the Thermoacidophilic Archaea
Kanehisa KOJOH, Hiroshi MATSUZAWA, and Takayoshi WAKAGI*
Department of Biotechnology, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113, Japan
The dicluster ferredoxins from thermoacidophilic archaea (1-9) are quite unique among those from other sources because of binding a zinc atom apart from the iron- sulfur clusters, as revealed by our X-ray chrystallographic analysis of Sulfolobus ferredoxin (1, 8, 9). The ferredoxin is composed of 103 amino acid residues, with a zinc atom at the interface of the two domains (a 37 residue N-terminal domain and a cluster-binding core domain). Zinc atom is suggested to stabilize the molecule (8, 9). To elucidate the role of the zinc atom as well as N-terminal extra sequence stretch, we prepared various mutants with partial or full deletion of the N-terminal domain, and investigated their stability.
Ferredoxin mutants with 0, 11, 16, 22, and 37 residues depleted from the N- terminal, designated as G1, V12, S17, G23, and V38, respectively, were prepared by PCR-mutagenesis methods. All the recombinant mutants were effectively expressed by the pET expression system (6) with two iron-sulfur clusters unaffected. Natural ferredoxin was prepared as previously reported (1). The stability of the iron-sulfur cluster was measured by monitoring the absorbance at 408 nm, at the temperature increasing from 50 to 100℃ with a rate of 1℃ / min. Contents of iron and zinc were determined by inductivity coupled plasma atomic emission spectrometry.
In natural ferredoxin, methylation of lysine-29 was detected by several criteria, while the recombinant ferredoxin G1 was not modified (7).
Natural, G1, and V12 ferredoxins bound 1 mol Zn/mol, while none was detected for S17, G23, or V38 mutants. The melting temperature (Tm) was above 100℃ for natural and G1, and 83℃ for V38 ferredoxin. Other deletion routants showed medium Tm values. Using the S17 mutant (Tm=89℃), concomitant degradation of secondary and tertially structure with cluster upon heat denaturation was observed by circular dichroism and fluorescence spectra analyses.
These results indicate that zinc atom and N-terminal extra stretch of the Sulfolobus ferredoxin are responsible for extreme thermostability of the molecule required for the organism to grow optimally at high tempearatures around 75-85℃.
1. Fujii, T., Moriyama, T., Takenaka, A., Tanaka, N., Wakagi, T., and Oshima, T. (1991) J. Biochem. (Tokyo), 110, 472-473.
2. Iwasaki, T., Wakagi, T., Isogai, Y.,Tanaka, K., Iizuka, T., and Oshima, T. (1994) J. Biol. Chem.,
