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

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, H_xO (x = 1, 2). Previous ¹⁷O ENDOR studies [Telser et al. (1986) J. Biol. Chem. 261, 4840–4846] had disclosed that H_x¹⁷O 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 Fe_a in substrate-free enzyme is a hydroxyl ion from the solvent; upon binding of substrate or substrate analogues at this Fe_a 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 Fe_a. 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.