Journal
JOURNAL OF BIOCHEMISTRY
Volume 142, Issue 5, Pages 577-586Publisher
OXFORD UNIV PRESS
DOI: 10.1093/jb/mvm163
Keywords
aquifex aeolicus; crystallization; iron-sulphur cluster; IscU; trimer
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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 angstrom 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 angstrom, where the asymmetric unit contains three molecules of Aa IscU. Successful crystallization of holo-Aa lscU-D38A strongly suggests that the trimer association carrying substoichiometric [2Fe-2S] cluster represents a conformationally stable oligomeric state.
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