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Originally published In Press as doi:10.1074/jbc.M201439200 on April 4, 2002

J. Biol. Chem., Vol. 277, Issue 24, 21397-21404, June 14, 2002
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Iron-Sulfur Cluster Biosynthesis
THERMATOGA MARITIMA IscU IS A STRUCTURED IRON-SULFUR CLUSTER ASSEMBLY PROTEIN*

Sheref S. MansyDagger §, Gong WuDagger , Kristene K. Surerus, and J. A. CowanDagger ||

From the Dagger  Evans Laboratory of Chemistry, Ohio State University, Columbus, Ohio 43210 and the  Department of Chemistry, University of Wisconsin, Milwaukee, Wisconsin 53201

Genetic evidence has indicated that Isc proteins play an important role in iron-sulfur cluster biogenesis. In particular, IscU is believed to serve as a scaffold for the assembly of a nascent iron-sulfur cluster that is subsequently delivered to target iron-sulfur apoproteins. We report the characterization of an IscU from Thermatoga maritima, an evolutionarily ancient hyperthermophilic bacterium. The stabilizing influence of a D40A substitution allowed characterization of the holoprotein. Mössbauer (delta  = 0.29 ± 0.03 mm/s, Delta EQ = 0.58 ± 0.03 mm/s), UV-visible absorption, and circular dichroism studies of the D40A protein show that T. maritima IscU coordinates a [2Fe-2S]2+ cluster. Thermal denaturation experiments demonstrate that T. maritima IscU is a thermally stable protein with a thermally unstable cluster. This is also the first IscU type domain that is demonstrated to possess a high degree of secondary and tertiary structure. CD spectra indicate 36.7% alpha -helix, 13.1% antiparallel beta -sheet, 11.3% parallel beta -sheet, 20.2% beta -turn, and 19.1% other at 20 °C, with negligible spectral change observed at 70 °C. Cluster coordination also has no effect on the secondary structure of the protein. The dispersion of signals in 1H-15N heteronuclear single quantum correlation NMR spectra of wild type and D40A IscU supports the presence of significant tertiary structure for the apoprotein, consistent with a scaffolding role, and is in marked contrast to other low molecular weight Fe-S proteins where cofactor coordination is found to be necessary for proper protein folding. Consistent with the observed sequence homology and proposed conservation of function for IscU-type proteins, we demonstrate T. maritima IscU-mediated reconstitution of human apoferredoxin.

* This work was supported in part by a grant from the Petroleum Research Fund, administered by the American Chemical Society (to J. A. C.), and by National Science Foundation Grant CHE-0111161 (to J. A. C.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ Supported by the National Institutes of Health Chemistry and Biology Interface Training Program at Ohio State University (Grant GM08512-03).

|| To whom correspondence should be addressed: Evans Laboratory of Chemistry, Ohio State University, 100 W. 18th Ave., Columbus, OH 43210. Fax: 614-292-1685; E-mail: cowan@chemistry.ohio-state.edu.

Copyright © 2002 by The American Society for Biochemistry and Molecular Biology, Inc.
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