Mode of Substrate Carboxyl Binding to the [4Fe-4S]+ Cluster of Reduced Aconitase As Studied by 170 and 13C Electron-Nuclear Doub

Proc. Natl. Acad. Sci. USA Vol. 84, pp. 8854-8858, December 1987 Biochemistry Mode of substrate carboxyl binding to the [4Fe-4S]+ cluster of reduced aconitase as studied by 170 and 13C electron-nuclear double resonance spectroscopy (aconitase mechanism/nitro analogues of substrate/isotopic labeling) MARY CLAIRE KENNEDY*, MELANIE WERSTt, JOSHUA TELSERt, MARK H. EMPTAGEt, HELMUT BEINERT*§, AND BRIAN M. HOFFMANt *Department of Biochemistry and 1National Biomedical ESR Center, Medical College of Wisconsin, Milwaukee, WI 53226; tDepartment of Chemistry, Northwestern University, Evanston, IL 60201; and tCentral Research and Development Department, E. I. duPont de Nemours, Wilmington, DE 19898 Contributed by Helmut Beinert, August 25, 1987 ABSTRACT The active form of aconitase has a diamag- ening of the EPR signal in the presence of 170-labeled netic [4Fe-4S]21 cluster. A specific iron ion (Fea, which is lost substrate$ and solvent water indicated that solvent HxO$ during inactivation) is the binding site for substrate, as shown and/or the -OH group of substrate could be bound to the by Mossbauer spectroscopy. We have studied the mode of [4Fe-4S]' cluster (3). The binding of HxO and -OH of an substrate and analogue binding at equilibrium to the paramag- analogue to substrate was demonstrated directly by subse- netic [4Fe-4S]+ cluster of the reduced active form by 170 and quent electron-nuclear double resonance (ENDOR) studies 13C electron-nuclear double resonance spectroscopy with spe- with the reduced enzyme (4). Samples equilibrated with cifically labeled substrates. The data show that with substrate, cis-aconitate in H2170 showed ENDOR signals from HO only the carboxyl at C-2 of the propane backbone is strongly bound to the cluster. No signal was seen that could be bound in addition to H20 or OH- (HxO) from the solvent, assigned to the exchangeable -OH of the substrate, which whereas in an isocitrate analogue that has a nitro group at C-2, suggested that substrate is bound to the enzyme largely in the the carboxyl and hydroxyl at C-1 are bound along with solvent form of cis-aconitate. However, the observation of 170 HxO. We conclude from these data that, on addition ofany one ENDOR resonances from active enzyme in the presence of ofthe three substrates, cis-aconitate is the predominant species C-1-170H labeled nitroisocitrate (1-hydroxy-2-nitro-1,3-pro- bound to Fe. of the cluster along with solvent HxO and that panedicarboxylate), an inhibitory reaction-intermediate an- cis-aconitate is bound in the citrate mode (carboxyl at C-2). The alogue (5), showed that the -OH group ofsubstrate can indeed results with the nitro analogue show that the enzyme can also be coordinated to the cluster. This complex of enzyme and bind a substrate-like ligand to the cluster in the alternative inhibitor also gave 170 ENDOR signals in H2170-enriched isocitrate mode (carboxyl at C-1), as is implicit in models solvent, which suggests that the cluster might simultaneously proposed for the aconitase reaction. coordinate the -OH of the substrate and HO of the solvent (4). The enzyme aconitase [citrate (isocitrate) hydro-lyase, EC 170 hyperfine line broadening was not observed when the 4.2.1.3] catalyzes the stereospecific interconversion of cit- three carboxyl groups of citrate were uniformly labeled with rate and isocitrate via the dehydrated intermediate cis-aconi- 170, which suggested that these groups do not, or at most tate, weakly, bind to the cluster (3). However, comparisons ofthe measured 170 broadening from the "70H-labeled inhibitor HO COO0 and H2170 with the broadening predicted from 170 hyperfine- COO- - H20 (a) COO + H2 interaction parameters obtained by ENDOR spectroscopy : - COO- - HO COO < (3) 0 revealed inherent limitations in the broadening measure- C00 + -COO- - H20 (Y) H20 ments (4). This indicated to us that the issue of carboxyl Citrate cis-Aconitate Isocitrate coordination could be resolved only by ENDOR examination of the ['70]carboxyl-labeled samples. Thus, we have indi- where the a and ,8 carbons are derived from oxaloacetate and vidually labeled the carboxyl groups ofcitrate with 170 or 13C the y carbon from acetyl-CoA. The active site of aconitase and performed ENDOR measurements. We find that despite contains a [4Fe-4S]2F cluster, which performs a catalytic the negative EPR results, the addition of carboxyl-labeled rather than an electron-transport function (1). The cluster citrate to the reduced active enzyme does indeed give 170 and loses one specific iron ion, designated Fea, during routine 13C ENDOR signals from a carboxyl group strongly coordi- purification to produce an inactive [3Fe-4S]+ structure. nated to the cluster. However, iron is readily reincorporated under reducing This observation fits well with a model for aconitase action conditions and activity is regained. The diamagnetic [4Fe- proposed by Gahan et al. (6) that involves carboxyl coordi- 4S]2+ cluster can be reduced to give the paramagnetic nation as a key element. In this model the intermediate is a [4Fe-4S]+ (S = 1/2) rhombic EPR state (gxyz = 2.06, 1.93, five-membered ring formed by Fea, the oxygen ofthe entering 1.86) that binds substrate strongly, with 30% retention of (or exiting) -OH, the carbon atom of cis-aconitate to which it activity (1, 2). is to be bound (a or,1), the carboxyl carbon attached to this Evidence was obtained by Mossbauer spectroscopy that carbon atom, and one of its carboxyl oxygens. Depending on substrate is bound to Fea in the 2+ and 1+ oxidation states. which of the two carboxyls of the oxaloacetate-derived Substrate binding to the 1+ form was shown by EPR to portion (a and 18) of substrate is bound to Fea, -OH binds to produce pronounced shifts in the g values. Hyperfine broad- Abbreviation: ENDOR, electron-nuclear double resonance. The publication costs of this article were defrayed in part by page charge $Unless specifically stated otherwise, substrate stands for either payment. This article must therefore be hereby marked "advertisement" citrate, isocitrate, cis-aconitate, or their mixtures. HxO stands for in accordance with 18 U.S.C. §1734 solely to indicate this fact. H20 or OH-, when they cannot be distinguished. 8854 Downloaded by guest on October 2, 2021 Biochemistry: Kennedy et al. Proc. Natl. Acad. Sci. USA 84 (1987) 8855 (is released from) C-1 (a) or C-2 (f3) of the propene backbone Table 1. 170 Labeling of Aconitase Substrates or Analogue of cis-aconitate, forming isocitrate or citrate, respectively Added* 170 Labeling (O)t Hyperfine Coupling (Fig. 1). Constant, Ay (MHz) METHODS Citrate HO 4JCOO- 0 All commercial chemicals and enzymes were obtained from Sigma and labeled compounds from ICON services (Summit, NJ). [1-17OH]Nitroisocitrate was kindly provided by J. V. HO {C 15 Schloss and [1,2,4,5-'3C]citrate by Jane Strouse (7). Aconi- tase was prepared and assayed as described (8). For spec- HO ECOO troscopy, aconitase was activated in 0.1 M Hepes (pH 7.5) 0 and desalted anaerobically on 1-ml columns of Sephadex Isocitrate G-50 that had been equilibrated with either 0.1 M Hepes (pH CO*- 7.5) (for photoreduction) or 0.1 M 3-[tris(hydroxymethyl) methylamino]-1-propanesulfonic acid (Taps, pH 8.5) (for Nitroisocitrate -N02 9, 13 dithionite reduction). Reduction to the 1+ state was carried out essentially as described (3). In all samples the concentration of substrate or inhibitor was 5-10 times the concen- -N02 qT tration of active enzyme, which was 0.25 ± 0.05 mM. ¶COO Labeling of Carboxyl Groups. [170]Carboxyl-labeled citric, *Note that in the presence of active aconitase any substrate malic and glyoxylic acids were prepared as described (3). The added is converted to an equilibrium mixture of the three individual carboxyls were labeled as shown in Table 1 and substrates. For the second entry an equilibrium mixture is actually added (see Methods). described below. Progress of the respective reactions-i.e., 3 formation of citrate or isocitrate-was followed enzymati- 'The a and carboxyls are derived from oxaloacetate, the y carboxyl from acetyl CoA. The a position corresponds to cally (9). C-I in nitroisocitrate. a and /3 carboxyls. [170]Carboxyl-labeled malate was +Taken from ref. 4. converted to citrate via the malate dehydrogenase and citrate synthase reactions (10). a carboxyl. KHCO3 (5 mg), trisodium phosphoenolpyruv- was used except with NaHl'3CO3 (99 atom %) instead of ate (2.5 mg), carbonic anhydrase (1 mg, 2500 units), and NaHCO3 and without CO2 gassing. MgCl2 (5 mM final concentration) were dissolved in 0.5 ml of y carboxyl. Isocitric lactone (174 mg) was dissolved in 0.25 H2170 (56.4 atom %) in a 1-ml vial. The gas space was filled ml of 12.5 M Na170H, which was prepared by adding metallic with C02, and the vial was closed and left to stand at 20°C for sodium to H2170 (51 atom %). The solution was heated at 30 min. Then dithiothreitol (10 ,ug), phosphoenolpyruvate 95°C for 30 min and diluted with 1 ml ofH2160, and trisodium carboxylase (2 mg, 2 units), and citrate synthase (20 ul, 25 isocitrate was converted to isocitric acid by passage through units) were added, followed by 15 mg of acetyl-CoA in four a column of AG 5OW-X4. The Iyophilized material was installments while the temperature was raised to 25°C. After dissolved in anhydrous 1 M HCI in diethyl ether and sealed 4 hr the tube was placed in a 90°C bath and the precipitated under an atmosphere of dry nitrogen. After 5 days the protein was collected by centrifugation. ether/HCI was evaporated under a stream ofdry nitrogen and the residue was hydrolyzed as above in 350 ,ul of 12.5 M 13 carboxyl.

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