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Mode of substrate carboxyl binding to the [4Fe-4S]+ cluster of reduced aconitase as studied by 17O and 13C electron-nuclear double resonance spectroscopy.
Proc Natl Acad Sci U S A. 1987 Dec; 84(24):8854-8.PN

Abstract

The active form of aconitase has a diamagnetic [4Fe-4S]2+ cluster. A specific iron ion (Fea, which is lost during inactivation) is the binding site for substrate, as shown by Mössbauer spectroscopy. We have studied the mode of substrate and analogue binding at equilibrium to the paramagnetic [4Fe-4S]+ cluster of the reduced active form by 17O and 13C electron-nuclear double resonance spectroscopy with specifically labeled substrates. The data show that with substrate, only the carboxyl at C-2 of the propane backbone is strongly bound in addition to H2O or OH- (HxO) from the solvent, whereas in an isocitrate analogue that has a nitro group at C-2, the carboxyl and hydroxyl at C-1 are bound along with solvent HxO. We conclude from these data that, on addition of any one of the three substrates, cis-aconitate is the predominant species bound to Fea of the cluster along with solvent HxO and that cis-aconitate is bound in the citrate mode (carboxyl at C-2). The results with the nitro analogue show that the enzyme can also bind a substrate-like ligand to the cluster in the alternative isocitrate mode (carboxyl at C-1), as is implicit in models proposed for the aconitase reaction.

Links

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Authors+Show Affiliations

Kennedy MC
Department of Biochemistry, Medical College of Wisconsin, Milwaukee 53226.
Werst M
No affiliation info available
Telser J
No affiliation info available
Emptage MH
No affiliation info available
Beinert H
No affiliation info available
Hoffman BM
No affiliation info available

MeSH

Aconitate HydrataseBinding SitesCitratesIron-Sulfur ProteinsMetalloproteinsOxygenSpectrum Analysis

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't
Research Support, U.S. Gov't, Non-P.H.S.
Research Support, U.S. Gov't, P.H.S.

Language

eng

PubMed ID

3480514

Citation

Kennedy, M C., et al. "Mode of Substrate Carboxyl Binding to the [4Fe-4S]+ Cluster of Reduced Aconitase as Studied By 17O and 13C Electron-nuclear Double Resonance Spectroscopy." Proceedings of the National Academy of Sciences of the United States of America, vol. 84, no. 24, 1987, pp. 8854-8.
Kennedy MC, Werst M, Telser J, et al. Mode of substrate carboxyl binding to the [4Fe-4S]+ cluster of reduced aconitase as studied by 17O and 13C electron-nuclear double resonance spectroscopy. Proc Natl Acad Sci U S A. 1987;84(24):8854-8.
Kennedy, M. C., Werst, M., Telser, J., Emptage, M. H., Beinert, H., & Hoffman, B. M. (1987). Mode of substrate carboxyl binding to the [4Fe-4S]+ cluster of reduced aconitase as studied by 17O and 13C electron-nuclear double resonance spectroscopy. Proceedings of the National Academy of Sciences of the United States of America, 84(24), 8854-8.
Kennedy MC, et al. Mode of Substrate Carboxyl Binding to the [4Fe-4S]+ Cluster of Reduced Aconitase as Studied By 17O and 13C Electron-nuclear Double Resonance Spectroscopy. Proc Natl Acad Sci U S A. 1987;84(24):8854-8. PubMed PMID: 3480514.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Mode of substrate carboxyl binding to the [4Fe-4S]+ cluster of reduced aconitase as studied by 17O and 13C electron-nuclear double resonance spectroscopy. AU - Kennedy,M C, AU - Werst,M, AU - Telser,J, AU - Emptage,M H, AU - Beinert,H, AU - Hoffman,B M, PY - 1987/12/1/pubmed PY - 1987/12/1/medline PY - 1987/12/1/entrez SP - 8854 EP - 8 JF - Proceedings of the National Academy of Sciences of the United States of America JO - Proc Natl Acad Sci U S A VL - 84 IS - 24 N2 - The active form of aconitase has a diamagnetic [4Fe-4S]2+ cluster. A specific iron ion (Fea, which is lost during inactivation) is the binding site for substrate, as shown by Mössbauer spectroscopy. We have studied the mode of substrate and analogue binding at equilibrium to the paramagnetic [4Fe-4S]+ cluster of the reduced active form by 17O and 13C electron-nuclear double resonance spectroscopy with specifically labeled substrates. The data show that with substrate, only the carboxyl at C-2 of the propane backbone is strongly bound in addition to H2O or OH- (HxO) from the solvent, whereas in an isocitrate analogue that has a nitro group at C-2, the carboxyl and hydroxyl at C-1 are bound along with solvent HxO. We conclude from these data that, on addition of any one of the three substrates, cis-aconitate is the predominant species bound to Fea of the cluster along with solvent HxO and that cis-aconitate is bound in the citrate mode (carboxyl at C-2). The results with the nitro analogue show that the enzyme can also bind a substrate-like ligand to the cluster in the alternative isocitrate mode (carboxyl at C-1), as is implicit in models proposed for the aconitase reaction. SN - 0027-8424 UR - https://www.unboundmedicine.com/medline/citation/3480514/Mode_of_substrate_carboxyl_binding_to_the_[4Fe_4S]+_cluster_of_reduced_aconitase_as_studied_by_17O_and_13C_electron_nuclear_double_resonance_spectroscopy_ DB - PRIME DP - Unbound Medicine ER -