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

• Published in: Biochemistry (Easton) 1990-11, Vol.29:46
• Main Authors: Werst, M.M., Hoffman, B.M., Kennedy, M.C., Beinert, H.
• Format: Article
• Language: English
• Subjects: 550201 - Biochemistry- Tracer Techniques; BARYONS; BASIC BIOLOGICAL SCIENCES; 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
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi00498a015

{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.