Linked Life Data

11939799

Source:http://linkedlifedata.com/resource/pubmed/id/11939799

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
15
pubmed:dateCreated
2002-4-9
pubmed:abstractText
Genetic studies of bacteria and eukaryotes have led to identification of several gene products that are involved in the biosynthesis of protein-bound iron-sulfur clusters. One of these proteins, ISU, is homologous to the N-terminus of bacterial NifU. The mature forms of His-tagged wild-type and D37A Schizosaccharomyces pombe ISU1 were cloned and overexpressed as inclusion bodies in Escherichia coli. The recombinant D37A protein was purified under denaturing conditions and subsequently reconstituted in vitro. By use of a 5-fold excess of iron and sulfide the reconstituted product was found to be red-brown in color, forming a homodimer of 17 kDa per subunit with approximately two iron atoms per monomer determined by protein and iron quantitation. UV-vis absorption and Mössbauer spectroscopies (delta = 0.29 +/- 0.05 mm/s; DeltaE(Q) = 0.59 +/- 0.05 mm/s) were used to characterize D37A ISU1 and show the presence of [2Fe-2S](2+) clusters in each subunit. Formation of the holo form of wild-type ISU1 was significantly less efficient using the same reconstitution conditions and is consistent with prior observations that the D37A substitution can stabilize protein-bound clusters. Relative to the human homologue, the yeast ISU is significantly less soluble at ambient temperatures. In both cases the native ISU1 is more sensitive to proton-mediated degradation relative to the D37A derivative. The lability of this family of proteins relative to [2Fe-2S] bearing ferredoxins most likely is of functional relevance for cluster transfer chemistry. Mössbauer parameters obtained for wild-type ISU1 (delta = 0.31 +/- 0.05 mm/s; DeltaE(Q) = 0.64 +/- 0.05 mm/s) were similar to those obtained for the D37A derivative. Cluster transfer from ISU1 to apo Fd is demonstrated: the first example of transfer from an ISU-type protein. A lower limit for k(2) of 80 M(-1) min(-1) was established for WT cluster transfer and a value of 18 M(-1) min(-1) for the D37A derivative. Finally, we have demonstrated through cross-linking studies that ferredoxin, an electron-transport protein, forms a complex with ISU1 in both apo and holo states. Cross-linking of holo ISU1 with holo Fd is consistent with a role for redox chemistry in cluster assembly and may mimic the intramolecular complex already defined in NifU.
pubmed:grant
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Apr
pubmed:issn
0006-2960
pubmed:author
pubmed:issnType
Print
pubmed:day
16
pubmed:volume
41
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
5024-32
pubmed:dateRevised
2009-11-19
pubmed:meshHeading
pubmed-meshheading:11939799-Amino Acid Sequence, pubmed-meshheading:11939799-Bacterial Proteins, pubmed-meshheading:11939799-Cloning, Molecular, pubmed-meshheading:11939799-DNA Primers, pubmed-meshheading:11939799-Fungal Proteins, pubmed-meshheading:11939799-Iron-Sulfur Proteins, pubmed-meshheading:11939799-Mitochondrial Proteins, pubmed-meshheading:11939799-Molecular Sequence Data, pubmed-meshheading:11939799-Mutagenesis, Site-Directed, pubmed-meshheading:11939799-Polymerase Chain Reaction, pubmed-meshheading:11939799-Protein Denaturation, pubmed-meshheading:11939799-Protein Subunits, pubmed-meshheading:11939799-Recombinant Proteins, pubmed-meshheading:11939799-Saccharomyces cerevisiae Proteins, pubmed-meshheading:11939799-Schizosaccharomyces, pubmed-meshheading:11939799-Sequence Alignment, pubmed-meshheading:11939799-Sequence Homology, Amino Acid
pubmed:year
2002
pubmed:articleTitle
Characterization of an iron-sulfur cluster assembly protein (ISU1) from Schizosaccharomyces pombe.
pubmed:affiliation
Evans Laboratory of Chemistry, The Ohio State University, 100 West 18th Avenue, Columbus, Ohio 43210, USA.
pubmed:publicationType
Journal Article, Comparative Study, Research Support, U.S. Gov't, P.H.S., Research Support, U.S. Gov't, Non-P.H.S., Research Support, Non-U.S. Gov't