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Mössbauer studies of aconitase. Substrate and inhibitor binding, reaction intermediates, and hyperfine interactions of reduced 3Fe and 4Fe clusters.
J Biol Chem. 1985 Jun 10; 260(11):6871-81.JB

Abstract

Active beef heart aconitase contains a [4Fe-4S] cluster. One iron of the cluster, Fea, is labile and can be removed easily by oxidation in air to yield the [3Fe-4S]1+ cluster of inactive aconitase. We have previously shown that substrate binds to Fea. We have continued our Mössbauer studies by further investigating the active and inactive forms of the enzyme. When active aconitase, [4Fe-4S]2+, is mixed with substrate, two species (substrates or intermediates bound to Fea) labeled S1 and S2 are obtained. With the nitroanalogs of citrate and isocitrate, thought to be transition state analogs, and fluorocitrate, species S2, but not S1, is observed, suggesting that S2 represents a carbanion transition state complex. We have prepared Mössbauer samples by rapid mix/rapid freeze techniques. Using either citrate, isocitrate or cis-aconitate, the natural substrates, we have been able to detect at 0 degree C reaction intermediates in the 5-35 ms time range and, studying enzyme substrate interactions at subzero temperatures in a water/methanol/ethylene glycol solvent, we have observed new species when substrates were added at -60 degrees C. Details of these experiments are given, although in neither case can unique interpretations be offered at this time. We have also investigated reduced active aconitase ([4Fe-4S]1+; EPR at g = 1.94) in the presence of substrate with material selectively enriched with 57Fe in either Fea or the other three cluster sites. The spectra were analyzed with a spin Hamiltonian, and the results are discussed and interpreted in terms of three inequivalent Fe sites in the cluster. Finally, we have studied enzyme containing the reduced [3Fe-4S]0 cluster. There is no indication that citrate binds to the 3Fe cluster, and since no significant activity was observed, we conclude that aconitase containing a 3Fe cluster is not active in either oxidation state.

Authors

Kent TA
No affiliation info available
Emptage MH
No affiliation info available
Merkle H
No affiliation info available
Kennedy MC
No affiliation info available
Beinert H
No affiliation info available
Münck E
No affiliation info available

MeSH

Aconitate HydrataseAnimalsCattleCitratesCitric AcidElectron Spin Resonance SpectroscopyIron-Sulfur ProteinsMetalloproteinsMyocardiumOxidation-ReductionSpectrum Analysis

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

2987236

Citation

Kent, T A., et al. "Mössbauer Studies of Aconitase. Substrate and Inhibitor Binding, Reaction Intermediates, and Hyperfine Interactions of Reduced 3Fe and 4Fe Clusters." The Journal of Biological Chemistry, vol. 260, no. 11, 1985, pp. 6871-81.
Kent TA, Emptage MH, Merkle H, et al. Mössbauer studies of aconitase. Substrate and inhibitor binding, reaction intermediates, and hyperfine interactions of reduced 3Fe and 4Fe clusters. J Biol Chem. 1985;260(11):6871-81.
Kent, T. A., Emptage, M. H., Merkle, H., Kennedy, M. C., Beinert, H., & Münck, E. (1985). Mössbauer studies of aconitase. Substrate and inhibitor binding, reaction intermediates, and hyperfine interactions of reduced 3Fe and 4Fe clusters. The Journal of Biological Chemistry, 260(11), 6871-81.
Kent TA, et al. Mössbauer Studies of Aconitase. Substrate and Inhibitor Binding, Reaction Intermediates, and Hyperfine Interactions of Reduced 3Fe and 4Fe Clusters. J Biol Chem. 1985 Jun 10;260(11):6871-81. PubMed PMID: 2987236.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Mössbauer studies of aconitase. Substrate and inhibitor binding, reaction intermediates, and hyperfine interactions of reduced 3Fe and 4Fe clusters. AU - Kent,T A, AU - Emptage,M H, AU - Merkle,H, AU - Kennedy,M C, AU - Beinert,H, AU - Münck,E, PY - 1985/6/10/pubmed PY - 1985/6/10/medline PY - 1985/6/10/entrez SP - 6871 EP - 81 JF - The Journal of biological chemistry JO - J Biol Chem VL - 260 IS - 11 N2 - Active beef heart aconitase contains a [4Fe-4S] cluster. One iron of the cluster, Fea, is labile and can be removed easily by oxidation in air to yield the [3Fe-4S]1+ cluster of inactive aconitase. We have previously shown that substrate binds to Fea. We have continued our Mössbauer studies by further investigating the active and inactive forms of the enzyme. When active aconitase, [4Fe-4S]2+, is mixed with substrate, two species (substrates or intermediates bound to Fea) labeled S1 and S2 are obtained. With the nitroanalogs of citrate and isocitrate, thought to be transition state analogs, and fluorocitrate, species S2, but not S1, is observed, suggesting that S2 represents a carbanion transition state complex. We have prepared Mössbauer samples by rapid mix/rapid freeze techniques. Using either citrate, isocitrate or cis-aconitate, the natural substrates, we have been able to detect at 0 degree C reaction intermediates in the 5-35 ms time range and, studying enzyme substrate interactions at subzero temperatures in a water/methanol/ethylene glycol solvent, we have observed new species when substrates were added at -60 degrees C. Details of these experiments are given, although in neither case can unique interpretations be offered at this time. We have also investigated reduced active aconitase ([4Fe-4S]1+; EPR at g = 1.94) in the presence of substrate with material selectively enriched with 57Fe in either Fea or the other three cluster sites. The spectra were analyzed with a spin Hamiltonian, and the results are discussed and interpreted in terms of three inequivalent Fe sites in the cluster. Finally, we have studied enzyme containing the reduced [3Fe-4S]0 cluster. There is no indication that citrate binds to the 3Fe cluster, and since no significant activity was observed, we conclude that aconitase containing a 3Fe cluster is not active in either oxidation state. SN - 0021-9258 UR - https://www.unboundmedicine.com/medline/citation/2987236/Mössbauer_studies_of_aconitase__Substrate_and_inhibitor_binding_reaction_intermediates_and_hyperfine_interactions_of_reduced_3Fe_and_4Fe_clusters_ DB - PRIME DP - Unbound Medicine ER -