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17O, 1H, and 2H electron nuclear double resonance characterization of solvent, substrate, and inhibitor binding to the [4Fe-4S]+ cluster of aconitase.
Biochemistry. 1990 Nov 20; 29(46):10526-32.B

Abstract

17O electron nuclear double resonance (ENDOR) studies at X-band (9-GHz) and Q-band (35-GHz) microwave frequencies reveal that the [4Fe-4S]+ cluster of substrate-free aconitase [citrate (isocitrate) hydro-lyase, EC 4.2.1.3] binds solvent, HxO (x = 1, 2). Previous 17O ENDOR studies [Telser et al. (1986) J. Biol. Chem. 261, 4840-4846] had disclosed that Hx17O binds to the enzyme-substrate complex and also to complexes of enzyme with the substrate analogues trans-aconitate and nitroisocitrate (1-hydroxy-2-nitro-1,3-propanedicarboxylate). We have used 1H and 2H ENDOR to characterize these solvent species. We propose that the fourth ligand of Fea in substrate-free enzyme is a hydroxyl ion from the solvent; upon binding of substrate or substrate analogues at this Fea site, the solvent species becomes protonated to form a water molecule. Previous 17O and 13C ENDOR studies [Kennedy et al. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 8854-8858] showed that only a single carboxyl, at C-2 of the propane backbone of cis-aconitate or at C-1 of the inhibitor nitroisocitrate, coordinates to the cluster. Together, these results imply that enzyme-catalyzed interconversion of citrate and isocitrate does not involve displacement of an endogenous fourth ligand, but rather addition of the anionic carboxylate ligand and a change in protonation state of a solvent species bound to Fea. We further report the 17O hyperfine tensor parameters of the C-2 carboxyl oxygen of substrate bound to the cluster as determined by the field dependence of the 17O ENDOR signals. 17O ENDOR studies also show that the carboxyl group of the inhibitor trans-aconitate binds similarly to that of substrate.

Authors+Show Affiliations

Werst MM
Department of Chemistry, Northwestern University, Evanston, Illinois 60208.
Kennedy MC
No affiliation info available
Beinert H
No affiliation info available
Hoffman BM
No affiliation info available

MeSH

Aconitate HydrataseDeuteriumElectron Spin Resonance SpectroscopyHydrogenIron-Sulfur ProteinsKineticsMagnetic Resonance SpectroscopyOxygen IsotopesProtein BindingSolvents

Pub Type(s)

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

Language

eng

PubMed ID

2176871

Citation

Werst, M M., et al. "17O, 1H, and 2H Electron Nuclear Double Resonance Characterization of Solvent, Substrate, and Inhibitor Binding to the [4Fe-4S]+ Cluster of Aconitase." Biochemistry, vol. 29, no. 46, 1990, pp. 10526-32.
Werst MM, Kennedy MC, Beinert H, et al. 17O, 1H, and 2H electron nuclear double resonance characterization of solvent, substrate, and inhibitor binding to the [4Fe-4S]+ cluster of aconitase. Biochemistry. 1990;29(46):10526-32.
Werst, M. M., Kennedy, M. C., Beinert, H., & Hoffman, B. M. (1990). 17O, 1H, and 2H electron nuclear double resonance characterization of solvent, substrate, and inhibitor binding to the [4Fe-4S]+ cluster of aconitase. Biochemistry, 29(46), 10526-32.
Werst MM, et al. 17O, 1H, and 2H Electron Nuclear Double Resonance Characterization of Solvent, Substrate, and Inhibitor Binding to the [4Fe-4S]+ Cluster of Aconitase. Biochemistry. 1990 Nov 20;29(46):10526-32. PubMed PMID: 2176871.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - 17O, 1H, and 2H electron nuclear double resonance characterization of solvent, substrate, and inhibitor binding to the [4Fe-4S]+ cluster of aconitase. AU - Werst,M M, AU - Kennedy,M C, AU - Beinert,H, AU - Hoffman,B M, PY - 1990/11/20/pubmed PY - 1990/11/20/medline PY - 1990/11/20/entrez SP - 10526 EP - 32 JF - Biochemistry JO - Biochemistry VL - 29 IS - 46 N2 - 17O electron nuclear double resonance (ENDOR) studies at X-band (9-GHz) and Q-band (35-GHz) microwave frequencies reveal that the [4Fe-4S]+ cluster of substrate-free aconitase [citrate (isocitrate) hydro-lyase, EC 4.2.1.3] binds solvent, HxO (x = 1, 2). Previous 17O ENDOR studies [Telser et al. (1986) J. Biol. Chem. 261, 4840-4846] had disclosed that Hx17O binds to the enzyme-substrate complex and also to complexes of enzyme with the substrate analogues trans-aconitate and nitroisocitrate (1-hydroxy-2-nitro-1,3-propanedicarboxylate). We have used 1H and 2H ENDOR to characterize these solvent species. We propose that the fourth ligand of Fea in substrate-free enzyme is a hydroxyl ion from the solvent; upon binding of substrate or substrate analogues at this Fea site, the solvent species becomes protonated to form a water molecule. Previous 17O and 13C ENDOR studies [Kennedy et al. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 8854-8858] showed that only a single carboxyl, at C-2 of the propane backbone of cis-aconitate or at C-1 of the inhibitor nitroisocitrate, coordinates to the cluster. Together, these results imply that enzyme-catalyzed interconversion of citrate and isocitrate does not involve displacement of an endogenous fourth ligand, but rather addition of the anionic carboxylate ligand and a change in protonation state of a solvent species bound to Fea. We further report the 17O hyperfine tensor parameters of the C-2 carboxyl oxygen of substrate bound to the cluster as determined by the field dependence of the 17O ENDOR signals. 17O ENDOR studies also show that the carboxyl group of the inhibitor trans-aconitate binds similarly to that of substrate. SN - 0006-2960 UR - https://www.unboundmedicine.com/medline/citation/2176871/17O_1H_and_2H_electron_nuclear_double_resonance_characterization_of_solvent_substrate_and_inhibitor_binding_to_the_[4Fe_4S]+_cluster_of_aconitase_ DB - PRIME DP - Unbound Medicine ER -