1m34: Difference between revisions

Revision as of 14:50, 21 February 2008

Nitrogenase Complex From Azotobacter Vinelandii Stabilized By ADP-Tetrafluoroaluminate

OverviewOverview

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.

About this StructureAbout this Structure

1M34 is a Protein complex structure of sequences from Azotobacter vinelandii with , , , , , , and as ligands. Active as Nitrogenase, with EC number 1.18.6.1 Full crystallographic information is available from OCA.

ReferenceReference

Biochemical and structural characterization of the cross-linked complex of nitrogenase: comparison to the ADP-AlF4(-)-stabilized structure., Schmid B, Einsle O, Chiu HJ, Willing A, Yoshida M, Howard JB, Rees DC, Biochemistry. 2002 Dec 31;41(52):15557-65. PMID:12501184

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-[[Image:1m34.gif|left|200px]]<br /><applet load="1m34" size="450" color="white" frame="true" align="right" spinBox="true"
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 caption="1m34, resolution 2.3&Aring;" />
 '''Nitrogenase Complex From Azotobacter Vinelandii Stabilized By ADP-Tetrafluoroaluminate'''<br />
 ==Overview==
-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.
+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.
 ==About this Structure==
-M34 is a [http://en.wikipedia.org/wiki/Protein_complex Protein complex] structure of sequences from [http://en.wikipedia.org/wiki/Azotobacter_vinelandii Azotobacter vinelandii] with CA, MG, ALF, HCA, CFM, CLF, SF4 and ADP as [http://en.wikipedia.org/wiki/ligands ligands]. Active as [http://en.wikipedia.org/wiki/Nitrogenase Nitrogenase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=1.18.6.1 1.18.6.1] Full crystallographic information is available from [http://ispc.weizmann.ac.il/oca-bin/ocashort?id=1M34 OCA].
+M34 is a [http://en.wikipedia.org/wiki/Protein_complex Protein complex] structure of sequences from [http://en.wikipedia.org/wiki/Azotobacter_vinelandii Azotobacter vinelandii] with <scene name='pdbligand=CA:'>CA</scene>, <scene name='pdbligand=MG:'>MG</scene>, <scene name='pdbligand=ALF:'>ALF</scene>, <scene name='pdbligand=HCA:'>HCA</scene>, <scene name='pdbligand=CFM:'>CFM</scene>, <scene name='pdbligand=CLF:'>CLF</scene>, <scene name='pdbligand=SF4:'>SF4</scene> and <scene name='pdbligand=ADP:'>ADP</scene> as [http://en.wikipedia.org/wiki/ligands ligands]. Active as [http://en.wikipedia.org/wiki/Nitrogenase Nitrogenase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=1.18.6.1 1.18.6.1] Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=1M34 OCA].
 ==Reference==
@@ Line 14: / Line 14: @@
 [[Category: Nitrogenase]]
 [[Category: Protein complex]]
-[[Category: Chiu, H.J.]]
+[[Category: Chiu, H J.]]
 [[Category: Einsle, O.]]
-[[Category: Howard, J.B.]]
+[[Category: Howard, J B.]]
-[[Category: Howard, J.B.Rees, D.C.]]
+[[Category: Howard, J B.Rees, D C.]]
 [[Category: Schmid, B.]]
 [[Category: Willing, A.]]
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 [[Category: signal transduction]]
-''Page seeded by [http://ispc.weizmann.ac.il/oca OCA ] on Tue Nov 20 21:06:31 2007''
+''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Thu Feb 21 13:50:58 2008''