57Fe, 33S, and l4N electron nuclear double resonance (ENDOR) studies have been performed to characterize the [4Fe-4S}+ cluster at the active site of aconitase. Q-band 57Fe ENDOR of isotopically enriched enzyme, both substrate free and in the enzyme–substrate complex, reveals four inequivalent iron sites. In agreement with Mössbauer studies [Kent et al. (1985) J. Biol. Chem. 260, 6371–6881], one of the iron ions, Fea, which is easily removed by oxidation to yield the [3Fe-4S]+ cluster of inactive aconitase, shows a dramatic change in the presence of substrate. The remaining iron sites, Feb1,2,3, show minor changes when substrate is bound. Methods devised by us for analyzing and simulating ENDOR spectra of a randomly oriented paramagnet have been used to determine the principal values and orientation relative to the g tensor for the hyperfine tensors of three of the four inequivalent iron sites of the [4Fe-4S]+ cluster, Fea, Feb2, and Feb3, in the substrate-free enzyme and the enzyme–substrate complex. The full tensor for the fourth site, Feb1, could not be obtained because its signal is seen only over a limited range of the EPR envelope. 33S ENDOR data for the enzyme–substrate complex using enzyme reconstituted with 33S show that the four inorganic bridging sulfide ions of the [4Fe-4S]+ cube have isotropic hyperfine couplings of A(S) < 12 MHz, and analysis indicates that they can be divided into two pairs, one with couplings of A(S1) ≲ 1 MHz and the other with A(S2) ~ 6–12 MHz; the analysis further places these pairs within the cube relative to the iron sites. 33S data for substrate-free enzyme is qualitatively similar and can be completely simulated by two types of S2−ion, with A(S1) ~ 7.5 and A(S2) ~ 9 MHz; the full hyperfine tensors have been determined. The hyperfine values for the two enzyme forms correspond to surprisingly small unpaired spin density on S2−. 14N ENDOR at Q-band reveals a nitrogen signal that does not change upon substrate binding.
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