Characterization of the (4Fe-4S) sup + cluster at the active site of aconitase by sup 57 Fe, sup 33 S, and sup 14 N electron nuclear double resonance spectrocopy

• Published in: Biochemistry (Easton) 1990-11, Vol.29:46
• Main Authors: Werst, M.M., Houseman, A.L.P., Hoffman, B.M., Kennedy, M.C., Beinert, H.
• Format: Article
• Language: English
• Subjects: 550201 - Biochemistry- Tracer Techniques; BASIC BIOLOGICAL SCIENCES; CARBON-OXYGEN LYASES; CHEMICAL STATE; ENDOR; ENZYME ACTIVITY; ENZYMES; EVEN-ODD NUCLEI; HYDRO-LYASES; HYPERFINE STRUCTURE; INTERMEDIATE MASS NUCLEI; IRON 57; IRON COMPOUNDS; IRON ISOTOPES; ISOTOPES; LIGHT NUCLEI; LYASES; MAGNETIC RESONANCE; METALLOPROTEINS; NITROGEN 14; NITROGEN ISOTOPES; NUCLEI; ODD-ODD NUCLEI; ORGANIC COMPOUNDS; PROTEINS; RESONANCE; STABLE ISOTOPES; SUBSTRATES; SULFUR 33; SULFUR ISOTOPES; TRANSITION ELEMENT COMPOUNDS
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi00498a016

{sup 57}Fe, {sup 33}S, and {sup 14}N electron nuclear double resonance (ENDOR) studies have been performed to characterize the (4Fe-4S){sup {plus}} cluster at the active site of aconitase. Q-band {sup 57}Fe ENDOR of isotopically enriched enzyme, both substrate free and in the enzyme-substrate complex, reveals four inequivalent iron sites. In agreement with Mossbauer studies, one of the iron ions, Fe{sub a}, which is easily removed by oxidation to yield the (3Fe-4S){sup {plus}} cluster of inactive aconitase, shows a dramatic change in the presence of substrate. The remaining iron sites, Fe{sub b1,2,3,} show minor changes when substrate is bound. Methods devised by the authors 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){sup {plus}} cluster, Fe{sub a}, Fe{sub b2}, and Fe{sub b3}, in the substrate-free enzyme and the enzyme-substrate complex. {sup 33}S ENDOR data for the enzyme-substrate complex using enzyme reconstituted with {sup 33}S show that the four inorganic bridging sulfide ions of the (4Fe-4S){sup {plus}} cube have isotropic hyperfine couplings of A(S) < 12 MHz, and analysis indicates that they can be divided into two pairs, the analysis further places these pairs within the cube relative to the iron sites. {sup 33}S data for substrate-free enzyme is qualitatively similar and can be completely simulated by two types of S{sup 2{minus}} ion, with A(S1) {approximately} 7.5 and A(S2) {approximately} 9 MHz; the full hyperfine tensors have been determined.