Oxygen-17, proton, and deuterium electron nuclear double resonance characterization of solvent, substrate, and inhibitor binding to the iron-sulfur [4Fe-4S]+ cluster of aconitase

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.

Spectroscopic Characterisation of an Aconitase (AcnA) of Escherichia coli

“…Mossbauer spectroscopy also indicated that the ligand environment of Fe, is significantly altered upon the addition of substrate (Emptage et al, 1983b). These findings are consistent with the indication from "0-ENDOR studies (Werst et al, 1990a) that substrate binds directly to the cluster, through Fe,, in this partially active form of the enzyme. Crystallographic studies have demonstrated that upon the addition of substrate (citrate, cis-aconitate or isocitrate) to the oxidised [4Fe-4SI2+ form Fe, forms an isocitrate complex with the substrate molecule binding via the Ca-hydroxyl oxygen atom and a Ca-carboxylate oxygen (denoted OHA and OA2, respectively;Lauble et al, 1992).…”

“…X-ray crystallography (Robbins and Stout, 1989) protein. Mossbauer (Kent et al, 1982; Emptage et al, 1983b) and electron-nuclear double resonance (ENDOR; Telser et al, 1986;Kennedy et al, 1987;Werst et al, 1990a) spectroscopic studies have revealed that Fe;, has, in addition to three inorganic sulphur bridging ligands, a hydroxyl anion ligand which persists but becomes protonated upon binding of the C2 carboxylate anion of cis-aconitate to Fe,. The inequivalence of the iron sites in the cluster has been highlighted by 57Fe-ENDOR and "S-ENDOR and it has been further shown that the nuclei of two of the inorganic sulphide bridging ligands are considerably more strongly coupled to the paramagnetic electron density in the reduced [4Fe-4S] ' cluster than are those of the other two (Werst et al, 1990b).…”

Nitric Oxide Sensitivity of the Aconitases

“…Aconitase was inactivated in extracts exposed to 1200 ppm NO ⅐ in N 2 (Fig. 5, line 2), and the virtual substrates fluorocitrate, trans-aconitate, and tricarballylate (29,40,41) completely blocked this inactivation (Fig. 5, compare lines 3-5 with lines 1 and 2).…”

“…Citrate 3-OH oxygen binding to Fe a may decrease the NO ⅐ reactivity, or alternatively, cis-aconitate may increase the reactivity by altering the ligand field of Fe a . The citrate hydroxyl oxygen does alter the Fe a in the mitochondrial aconitase as indicated by ENDOR (electron nuclear double resonance), EPR, and Möss-bauer spectroscopy (40,41,44). However, the ability of the hydroxylless trans-aconitate and tricarballylate to protect aconitase equally with that of the hydroxyl-containing inhibitor fluorocitrate (Fig.…”

Electron-nuclear double resonance spectroscopy (and electron spin-echo envelope modulation spectroscopy) in bioinorganic chemistry

“…A key mechanistic question regarding this family is the role of the cluster in this process. In particular, the [4Fe-4S] cluster of enzymes in the Fe-S͞ AdoMet family has a ''unique'' iron site that is not coordinated to the enzyme by a cysteinyl sulfur; does this Fe have a catalytic function, as is the case for aconitase (25)?…”