Characterization and crystallization of an IscU-type scaffold protein with bound [2Fe-2S] cluster from the hyperthermophile, aquifex aeolicus.
IscU plays a key role during iron-sulphur (Fe-S) cluster biosynthesis as a scaffold for the assembly of a nascent, highly labile Fe-S cluster. Here we report the characterization of an IscU-type protein (Aa IscU) from the hyperthermophilic bacterium Aquifex aeolicus. Unlike other known homologues of IscU, expression of Aa IscU in Escherichia coli has yielded an Fe-S cluster-containing holo-protein. Biochemical and spectroscopic studies of the wild-type Aa IscU and its Asp38-to-Ala substituted (D38A) variant molecule indicate that the holo-protein forms a trimer containing substoichiometric [2Fe-2S] cluster with its stability substantially increased by a D38A substitution. The [2Fe-2S] cluster was oxygen-labile and upon loss of the cluster, the resultant apo-form dissociated into a smaller species, a mixture of monomer and dimer with the dimer form predominating. Reddish-brown crystals of holo-Aa IscU-D38A were obtained under anaerobic conditions, that gave diffractions beyond 2.0 A resolution with synchrotron radiation. The crystal belongs to the space group P2(1)2(1)2 with unit-cell parameters a = 72.6, b = 122.3, c = 62.4 A, where the asymmetric unit contains three molecules of Aa IscU. Successful crystallization of holo-Aa IscU-D38A strongly suggests that the trimer association carrying substoichiometric [2Fe-2S] cluster represents a conformationally stable oligomeric state.
Shimomura
Yoshimitsu
Y
Department of Biological Sciences, Graduate School of Science, Osaka University, Toyonaka, Osaka 560-0043, Japan.
Kamikubo
Hironari
H
Nishi
Yoshinori
Y
Masako
Takuya
T
Kataoka
Mikio
M
Kobayashi
Yuji
Y
Fukuyama
Keiichi
K
Takahashi
Yasuhiro
Y
D000595:Amino Acid Sequence; D001419:Bacteria / Q000737:chemistry* / Q000378:metabolism; D003460:Crystallization; D004591:Electrophoresis, Polyacrylamide Gel; D007506:Iron-Sulfur Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D008969:Molecular Sequence Data; D012542:Scattering, Radiation; D013056:Spectrophotometry, Ultraviolet; D017356:Synchrotrons; D014965:X-Rays
D007506:Iron-Sulfur Proteins; C523690:IscU protein, mouse