17O, 1H, and 2H Electron Nuclear Double Resonance Characterization of Solvent, Substrate, and Inhibitor Binding to the [4Fe-4S]+ Cluster of Aconitase

Melanie M. Werst, Brian M. Hoffman, Mary Claire Kennedy, Helmut Beinert*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

77 Scopus citations

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.

Original languageEnglish (US)
Pages (from-to)10526-10532
Number of pages7
JournalBiochemistry
Volume29
Issue number46
DOIs
StatePublished - Nov 1 1990

ASJC Scopus subject areas

  • Biochemistry

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