Journal Article

Characterization and Crystallization of an IscU-type Scaffold Protein with Bound [2Fe–2S] Cluster from the Hyperthermophile, <i>Aquifex aeolicus</i>

Yoshimitsu Shimomura, Hironari Kamikubo, Yoshinori Nishi, Takuya Masako, Mikio Kataoka, Yuji Kobayashi, Keiichi Fukuyama and Yasuhiro Takahashi

in The Journal of Biochemistry

Published on behalf of The Japanese Biochemical Society

Volume 142, issue 5, pages 577-586
Published in print November 2007 | ISSN: 0021-924X
Published online September 2007 | e-ISSN: 1756-2651 | DOI: http://dx.doi.org/10.1093/jb/mvm163
Characterization and Crystallization of an IscU-type Scaffold Protein with Bound [2Fe–2S] Cluster from the Hyperthermophile, Aquifex aeolicus

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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 Å resolution with synchrotron radiation. The crystal belongs to the space group P21212 with unit-cell parameters a = 72.6, b = 122.3, c = 62.4 Å, 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.

Keywords: Aquifex aeolicus; crystallization; iron–sulphur cluster; IscU; trimer

Journal Article.  5762 words.  Illustrated.

Subjects: Biochemistry

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