Linked Life Data

12501184

Source:http://linkedlifedata.com/resource/pubmed/id/12501184

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
52
pubmed:dateCreated
2002-12-26
pubmed:databankReference
pubmed:abstractText
The transient formation of a complex between the component Fe- and MoFe-proteins of nitrogenase represents a central event in the substrate reduction mechanism of this enzyme. Previously, we have isolated an N-[3-(dimethylamino)propyl]-N'-ethylcarbodiimide (EDC) cross-linked complex of these proteins stabilized by a covalent isopeptide linkage between Glu 112 and Lys beta400 of the Fe-protein and MoFe-protein, respectively [Willing, A., et al. (1989) J. Biol. Chem. 264, 8499-8503; Willing, A., and Howard, J. B. (1990) J. Biol. Chem. 265, 6596-6599]. We report here the biochemical and structural characterization of the cross-linked complex to assess the mechanistic relevance of this species. Glycinamide inhibits the cross-linking reaction, and is found to be specifically incorporated into Glu 112 of the Fe-protein, without detectable modification of either of the neighboring residues (Glu 110 and Glu 111). This modified protein is still competent for substrate reduction, demonstrating that formation of the cross-linked complex is responsible for the enzymatic inactivation, and not the EDC reaction or the modification of the Fe-protein. Crystallographic analysis of the EDC-cross-linked complex at 3.2 A resolution confirms the site of the isopeptide linkage. The nature of the protein surfaces around the cross-linking site suggests there is a strong electrostatic component to the formation of the complex, although the interface area between the component proteins is small. The binding footprints between proteins in the cross-linked complex are adjacent, but with little overlap, to those observed in the complex of the nitrogenase proteins stabilized by ADP-AlF(4)(-). The results of these studies suggest that EDC cross-linking traps a nucleotide-independent precomplex of the nitrogenase proteins driven by complementary electrostatic interactions that subsequently rearranges in a nucleotide-dependent fashion to the electron transfer competent state observed in the ADP-AlF(4)(-) structure.
pubmed:grant
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
http://linkedlifedata.com/resource/pubmed/chemical/Adenosine Diphosphate, http://linkedlifedata.com/resource/pubmed/chemical/Aluminum Compounds, http://linkedlifedata.com/resource/pubmed/chemical/Cross-Linking Reagents, http://linkedlifedata.com/resource/pubmed/chemical/Ethyldimethylaminopropyl..., http://linkedlifedata.com/resource/pubmed/chemical/Fluorides, http://linkedlifedata.com/resource/pubmed/chemical/Glycine, http://linkedlifedata.com/resource/pubmed/chemical/Molybdoferredoxin, http://linkedlifedata.com/resource/pubmed/chemical/Multienzyme Complexes, http://linkedlifedata.com/resource/pubmed/chemical/Nitrogenase, http://linkedlifedata.com/resource/pubmed/chemical/Nonheme Iron Proteins, http://linkedlifedata.com/resource/pubmed/chemical/glycine amide, http://linkedlifedata.com/resource/pubmed/chemical/tetrafluoroaluminate
pubmed:status
MEDLINE
pubmed:month
Dec
pubmed:issn
0006-2960
pubmed:author
pubmed:issnType
Print
pubmed:day
31
pubmed:volume
41
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
15557-65
pubmed:dateRevised
2008-11-21
pubmed:meshHeading
pubmed:year
2002
pubmed:articleTitle
Biochemical and structural characterization of the cross-linked complex of nitrogenase: comparison to the ADP-AlF4(-)-stabilized structure.
pubmed:affiliation
Division of Chemistry and Chemical Engineering 114-96, California Institute of Technology, Pasadena, CA 91125, USA.
pubmed:publicationType
Journal Article, 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