Characterization of the [4Fe-4S]+ Cluster at the Active Site of Aconitase by 57Fe, 33S, and 14N Electron Nuclear Double Resonance Spectroscopy

Melanie M. Werst, Andrew L.P. Houseman, Brian M. Hoffman, Mary Claire Kennedy, Helmut Beinert*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

60 Scopus citations


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.

Original languageEnglish (US)
Pages (from-to)10533-10540
Number of pages8
Issue number46
StatePublished - Nov 1 1990

ASJC Scopus subject areas

  • Biochemistry


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