The crystal structures of mitochondrial aconitase with isocitrate and nitroisocitrate bound have been solved and refined to R factors of 0.179 and 0.161, respectively, for all observed data in the range 8.0-2.1 A. Porcine heart enzyme was used for determining the structure with isocitrate bound. The presence of isocitrate in the crystals was corroborated by Mössbauer spectroscopy. Bovine heart enzyme was used for determining the structure with the reaction intermediate analogue nitroisocitrate bound. The inhibitor binds to the enzyme in a manner virtually identical to that of isocitrate. Both compounds bind to the unique Fe atom of the [4Fe-4S] cluster via a hydroxyl oxygen and one carboxyl oxygen. A H2O molecule is also bound, making Fe six-coordinate. The unique Fe is pulled away approximately 0.2 A from the corner of the cubane compared to the position it would occupy in a symmetrically ligated [4Fe-4S] cluster. At least 23 residues from all four domains of aconitase contribute to the active site. These residues participate in substrate recognition (Arg447, Arg452, Arg580, Arg644, Gln72, Ser166, Ser643), cluster ligation and interaction (Cys358, Cys421, Cys424, Asn258, Asn446), and hydrogen bonds supporting active site side chains (Ala74, Asp568, Ser571, Thr567). Residues implicated in catalysis are Ser642 and three histidine-carboxylate pairs (Asp100-His101, Asp165-His147, Glu262-His167). The base necessary for proton abstraction from C beta of isocitrate appears to be Ser642; the O gamma atom is proximal to the calculated hydrogen position, while the environment of O gamma suggests stabilization of an alkoxide (an oxyanion hole formed by the amide and side chain of Arg644). The histidine-carboxylate pairs appear to be required for proton transfer reactions involving two oxygens bound to Fe, one derived from solvent (bound H2O) and one derived from substrate hydroxyl. Each oxygen is in contact with a histidine, and both are in contact with the side chain of Asp165, which bridges the two sites on the six-coordinate Fe.

It has been known for many years that f luoroacetate and f luorocitrate when metabolized are highly toxic, and that at least one effect of f luorocitrate is to inactivate aconitase. In this paper we present evidence supporting the hypothesis that the (؊)-erythro diastereomer of 2-f luorocitrate acts as a mechanism based inhibitor of aconitase by first being converted to f luoro-cis-aconitate, followed by addition of hydroxide and with loss of f luoride to form 4-hydroxy-trans-aconitate (HTn), which binds very tightly, but not covalently, to the enzyme. Formation of HTn by these reactions is in accord with the working model for the enzyme mechanism. That HTn is the product of f luorocitrate inhibition is supported by the crystal structure of the enzymeinhibitor complex at 2.05-Å resolution, release of f luoride stoichiometric with total enzyme when (؊)-erythro-2-f luorocitrate is added, HPLC analysis of the product, slow displacement of HTn by 10 6 -fold excess of isocitrate, and previously published Mössbauer experiments. When (؉)-erythro-2-f luorocitrate is added to aconitase, the release of f luoride is stoichiometric with total substrate added, and HPLC analysis of the products indicates the formation of oxalosuccinate, and its derivative ␣-ketoglutarate. This is consistent with the proposed mechanism, as is the formation of HTn from (؊)-erythro-2-f luorocitrate. The structure of the inhibited complex reveals that HTn binds like the inhibitor trans-aconitate while providing all the interactions of the natural substrate, isocitrate. The structure exhibits four hydrogen bonds <2.7 Å in length involving HTn, H 2 O bound to the [4Fe-4S] cluster, Asp-165 and His-167, as well as low temperature factors for these moieties, consistent with the observed very tight binding of the inhibitor.The mechanism of the inhibitory effects of fluorocitrate on the enzyme aconitase [citrate(isocitrate)hydrolyase, EC 4.2.1.3] has been a long-standing problem in biochemistry. The toxic nature of fluoroacetate was discovered over 50 years ago (1, 2) and citrate was found to accumulate in tissues poisoned with compounds that could provide the fluoroacetyl residue. On this basis Peters (3) and Martius (4) proposed that the inhibitory substance was a fluorotricarboxylic acid. Subsequently it was shown that 2-fluorocitrate is indeed produced metabolically via the citrate synthase reaction (5) and that in the presence of this substance the enzyme aconitase is inhibited (6). Aconitase catalyzes the conversion of citrate to isocitrate (Iso) via the obligatory intermediate cis-aconitate (Scheme I).