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Journal of Biochemistry Advance Access originally published online on September 10, 2007
Journal of Biochemistry 2007 142(5):577-586; doi:10.1093/jb/mvm163
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© 2007 The Japanese Biochemical Society

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

Yoshimitsu Shimomura1, Hironari Kamikubo2, Yoshinori Nishi3, Takuya Masako1, Mikio Kataoka2, Yuji Kobayashi3, Keiichi Fukuyama1 and Yasuhiro Takahashi1,*

1Department of Biological Sciences, Graduate School of Science, Osaka University, Toyonaka, Osaka 560-0043; 2Graduate School of Materials Science, Nara Institute of Science and Technology (NAIST), Ikoma, Nara 630-0192; and 3Osaka University of Pharmaceutical Sciences, Takatsuki, Osaka, 569-1094, Japan

*To whom correspondence should be addressed. Tel: +81-6-6850-5423, Fax: +81-6-6850-5425, E-mail: ytaka{at}bio.sci.osaka-u.ac.jp

Received July 5, 2007; Accepted August 5, 2007


  Abstract

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.

Key Words: Aquifex aeolicus, crystallization, iron–sulphur cluster, IscU, trimer

Abbreviations: Fe–S, iron–sulphur; SAXS, small-angle X-ray scattering; WT, wild-type


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