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Biochemical and Structural Characterization of the Cross-linked Complex of Nitrogenase: Comparison to the ADP-AIF_4^--Stabilized Structure

Schmid, Benedikt and Einsle, Oliver and Chiu, Hsui-Ju and Willing, Andreas and Yoshida, Mika and Howard, James B. and Rees, Douglas C. (2002) Biochemical and Structural Characterization of the Cross-linked Complex of Nitrogenase: Comparison to the ADP-AIF_4^--Stabilized Structure. Biochemistry, 41 (52). pp. 15557-15565. ISSN 0006-2960. https://resolver.caltech.edu/CaltechAUTHORS:20141212-145725733

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Abstract

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 β400 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 Å 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.


Item Type:Article
Related URLs:
URLURL TypeDescription
http://dx.doi.org/10.1021/bi026642bDOIArticle
http://pubs.acs.org/doi/abs/10.1021/bi026642bPublisherArticle
ORCID:
AuthorORCID
Rees, Douglas C.0000-0003-4073-1185
Contact Email Address:phoebe@caltech.edu
Additional Information:Copyright © 2002 American Chemical Society. Received August 15, 2002. Revised Manuscript Received October 27, 2002. We thank A. Deacon and the staff at the Stanford Synchrotron Radiation Laboratory for assistance with data collection.
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Funding AgencyGrant Number
Howard Hughes Medical Institute (HHMI)UNSPECIFIED
Issue or Number:52
Record Number:CaltechAUTHORS:20141212-145725733
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:20141212-145725733
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:52790
Collection:CaltechAUTHORS
Deposited By: SWORD User
Deposited On:22 Dec 2014 19:42
Last Modified:03 Oct 2019 07:44

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