Zinc and an N-terminal extra stretch of the ferredoxin from a thermoacidophilic archaeon stabilize the molecule at high temperature.
Ferredoxin from the thermoacidophilic archaeon Sulfolobus sp. strain 7 has a 36-residue extra domain at its N-terminus and a 67-residue core domain carrying two iron-sulfur clusters. A zinc ion is held at the interface of the two domains through tetrahedral coordination of three histidine residues (-6, -19 and -34) and one aspartic acid residue (-76) [Fujii, T., Hata, Y., Oozeki, M., Moriyama, H., Wakagi, T., Tanaka, N. & Oshima, T. (1997) Biochemistry 36, 1505-1513]. To elucidate the roles of the novel zinc ion and the extra N-terminal domain, a series of truncated mutants was constructed: G1, V12, S17, G23, L31 and V38, which lack residues 0, 11, 16, 22, 30 and 37 starting from the N-terminus, respectively. A mutant with two histidine residues each replaced by an alanine residue, H16A/H19A, was also constructed. All the mutant ferredoxins had two iron-sulfur clusters, while zinc was retained only in G1 and V12. The thermal stability of the proteins was investigated by monitoring A408; the melting temperature (Tm) was approximately 109 degrees C for the natural ferredoxin, approximately 109 degrees C for G1, 97.6 degrees C for V12, 89.0 degrees C for S17, 89.2 degrees C for G23, 89.3 degrees C for L31, 82.1 degrees C for V38, and 89.4 degrees C for H16A/H19A. Km and Vmax values of 2-oxoglutarate:ferredoxin oxidoreductase for natural ferredoxin, G1, S17 and L31 were similar, suggesting that electron-accepting activities were not affected by the deletion. The combination of CD and fluorescent spectroscopic analyses with truncated mutant S17 indicated that not only the clusters but also the secondary and tertiary structures were simultaneously degraded at a Tm around 89 degrees C. These results unequivocally demonstrate that the zinc ion and certain parts, but not all, of the extra sequence stretch in the N-terminal domain are responsible not for function but for thermal stabilization of the molecule.
Kojoh
K
K
Department of Biotechnology, University of Tokyo, Japan.
Matsuzawa
H
H
Wakagi
T
T
D000595:Amino Acid Sequence; D001483:Base Sequence; D017931:DNA Primers; D005288:Ferredoxins / Q000737:chemistry*; D006358:Hot Temperature; D011487:Protein Conformation; D011489:Protein Denaturation; D016944:Sulfolobus / Q000737:chemistry*; D015032:Zinc / Q000737:chemistry*
D017931:DNA Primers; D005288:Ferredoxins; D015032:Zinc