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

Journal Article · Thu Nov 01 04:00:00 EST 1990 · Biochemistry; (USA)

DOI:https://doi.org/10.1021/bi00498a016· OSTI ID:5527381

Werst, M M; Houseman, A L.P.; Hoffman, B M ^[1]; Kennedy, M C; Beinert, H ^[2]

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1. Northwestern Univ., Evanston, IL (USA)
2. Medical College of Wisconsin, Milwaukee (USA)

{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.

• OSTI ID: 5527381
• Journal Information: Biochemistry; (USA), Journal Name: Biochemistry; (USA) Vol. 29:46; ISSN 0006-2960; ISSN BICHA
• Country of Publication: United States
• Language: English

Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 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