Characterization of the iron-sulfur [4Fe-4S]+ cluster at the active site of aconitase by iron-57, sulfur-33, and nitrogen-14 electron nuclear double resonance spectroscopy

Abstract

57Fe, 33S, and 14N electron nuclear double resonance (ENDOR) studies have been performed to characterize the [4Fe-4S]+ cluster at the active site of aconitase. Q-band 57Fe ENDOLR 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) less than 12 MHz, and analysis indicates that they can be divided into two pairs, one with couplings of A(S1) approximately less than 1 MHz and the other with A(S2) approximately 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) approximately 7.5 and A(S2) approximately 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.

13C and 63,65Cu ENDOR studies of CO Dehydrogenase from Oligotropha carboxidovorans. Experimental Evidence in Support of a Copper–Carbonyl Intermediate

“…13 CO signals are identified by comparing centers prepared with 12,13 CO, and the [2Fe-2S] centers give no ENDOR response in the vicinity of 63,65 Cu ENDOR transitions. 20–23 …”

The CCG-domain-containing subunit SdhE of succinate:quinone oxidoreductase from Sulfolobus solfataricus P2 binds a [4Fe–4S] cluster

“…Incubation with 1 mM potassium ferricyanide (E°’ = +356 mV) resulted in an irreversible degradation of the cluster. Since the cluster was stable under air (E°’ = +818 mV) this degradation is probably caused by the complexation of iron by ferricyanide rather than by the oxidizing conditions [26]. In redox titrations at potentials higher than -50 mV a second signal in the g = 1.992 region became detectable which overlapped with the g zyx = 2.017, 2.008 and 1.947 signal (Fig.…”

Characterization of MOCS1A, an Oxygen-sensitive Iron-Sulfur Protein Involved in Human Molybdenum Cofactor Biosynthesis

“…The paradigm here is aconitase in which the active site comprises a [4Fe-4S] cluster ligated by only three cysteines with the non-cysteinyl-ligated unique iron site involved in the binding and activation of the substrate (72,73). The formation of precursor Z involves a rearrangement reaction of the carbon atom at the 8-position of 5Ј-GTP (5, 6).…”