sup 17 O, sup 1 H, and sup 2 H electron nuclear double resonance characterization of solvent, substrate, and inhibitor binding to the (4Fe-4S) sup + cluster of aconitase

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

DOI:https://doi.org/10.1021/bi00498a015· OSTI ID:5605294

Werst, M M; Hoffman, B M ^[1]; Kennedy, M C; Beinert, H ^[2]

---

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

{sup 17}O electron nuclear double resonance (ENDOR) studies at X-band (9-GHz) and Q-band (35-GHz) microwave frequencies reveal that the (4Fe-4S){sup {plus}} cluster of substrate-free aconitase (citrate (isocitrate) hydro-lyase, EC 4.2.1.3) binds solvent, H{sub x}O (x = 1,2). Previous {sup 17}O ENDOR studies had disclosed that H{sub x}{sup 17}O binds to the enzyme-substrate complex and also to complexes of enzyme with the substrate analogues trans-aconitate and nitroisocitrate (1-hydroxy-2-nitro-1,3-propanedicarboxylate). The authors have used {sup 1}H and {sup 2}H ENDOR to characterize these solvent species. The authors propose that the fourth ligand of Fe{sub a} in substrate-free enzyme is a hydroxyl ion from the solvent; upon binding of substrate or substrate analogues at this Fe{sub a} site, the solvent species becomes protonated to form a water molecule. Previous {sup 17}O and {sup 13}C ENDOR studies showed that only a single carboxyl, at C-2 of the propane backbone of cis-aconitate or at C-1 of the inhibitor nitroisocitrate, coordinates to the cluster. Together, these results imply that enzyme-catalyzed interconversion of citrate and isocitrate does not involve displacement of an endogenous fourth ligand, but rather addition of the anionic carboxylate ligand and a change in protonation state of a solvent species bound to Fe{sub a}. The authors further report the {sup 17}O hyperfine tensor parameters of the C-2 carboxyl oxygen of substrate bound to the cluster as determined by the field dependence of the {sup 17}O ENDOR signals. {sup 17}O ENDOR studies also show that the carboxyl group of the inhibitor trans-aconitate binds similarly to that off substrate.

• OSTI ID: 5605294
• 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
BARYONS
CARBON-OXYGEN LYASES
CARBOXYLIC ACID SALTS
CHEMICAL REACTIONS
CITRATES
CROSS-LINKING
DEUTERIUM COMPOUNDS
ELECTROMAGNETIC RADIATION
ELEMENTARY PARTICLES
ENDOR
ENZYMES
EVEN-ODD NUCLEI
FERMIONS
HADRONS
HYDRO-LYASES
HYDROGEN COMPOUNDS
HYPERFINE STRUCTURE
ISOTOPES
LIGHT NUCLEI
LYASES
MAGNETIC RESONANCE
MICROWAVE RADIATION
NUCLEI
NUCLEONS
OXYGEN 17
OXYGEN ISOTOPES
POLYMERIZATION
PROTONS
RADIATIONS
RESONANCE
SOLVENTS
STABLE ISOTOPES
SUBSTRATES