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
- Northwestern Univ., Evanston, IL (USA)
- 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), Vol. 29:46; ISSN 0006-2960
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
HYDRO-LYASES
ENDOR
SUBSTRATES
CROSS-LINKING
CITRATES
DEUTERIUM COMPOUNDS
HYPERFINE STRUCTURE
MICROWAVE RADIATION
OXYGEN 17
PROTONS
SOLVENTS
BARYONS
CARBON-OXYGEN LYASES
CARBOXYLIC ACID SALTS
CHEMICAL REACTIONS
ELECTROMAGNETIC RADIATION
ELEMENTARY PARTICLES
ENZYMES
EVEN-ODD NUCLEI
FERMIONS
HADRONS
HYDROGEN COMPOUNDS
ISOTOPES
LIGHT NUCLEI
LYASES
MAGNETIC RESONANCE
NUCLEI
NUCLEONS
OXYGEN ISOTOPES
POLYMERIZATION
RADIATIONS
RESONANCE
STABLE ISOTOPES
550201* - Biochemistry- Tracer Techniques