Iron-Sulfur Cluster Biosynthesis

Sheref S. Mansy, Gong Wu, Kristene K. Surerus, J. A. Cowan
2002 Journal of Biological Chemistry  
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. Mö
more » ... bauer (␦ ‫؍‬ 0.29 ؎ 0.03 mm/s, ⌬E Q ‫؍‬ 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% ␣-helix, 13.1% antiparallel ␤-sheet, 11.3% parallel ␤-sheet, 20.2% ␤-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 1 H-15 N 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.
doi:10.1074/jbc.m201439200 pmid:11934893 fatcat:tekrs6zazja6dnh5qmsskarvku