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Concurrent Reduction of Iodine and Oxidation of EDTA at the Active Site of Horseradish Peroxidase: Probing the Iodine Binding Site by Optical Difference Spectroscopy and Steady State Kinetic Analysis for the Formation of Active Enzyme-If -EDTA Ternary Complex for Iodine Reductase Activity

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Title Concurrent Reduction of Iodine and Oxidation of EDTA at the Active Site of
Horseradish Peroxidase: Probing the Iodine Binding Site by Optical Difference
Spectroscopy and Steady State Kinetic Analysis for the Formation of Active Enzyme-If -EDTA Ternary Complex for Iodine Reductase Activity
 
Creator Adak, Subrata
Bhattacharya, Dipak Kr.
Majumder, Avijit
Bandyopadhyay, Uday
Banerjee, Ranajit K
 
Subject Structural Biology & Bioinformatics
 
Description Horseradish peroxidase (HRP) catalyzes the reduction of iodine to iodide by EDTA with
pseudocatalatic degradation of H202 to 0 2 (Banerjee et al., (1986) J. Biol. Chem. 261, 10592-10597;
and Banerjee (1989) J. Biol. Chem. 264, 9188-9194). The reduction of iodine (If) is dependent on
EDTA concentration and is blocked by spin trap, DMPO, indicating the involvement of free radical species
in the reduction process. Incubation of EDTA with both HRP and H202 results in the appearance of
triplet ESR signal of spin-trapped EDTA radical (aN = 15 G), indicating its one-electron oxidation to a
nitrogen-centered monocation radical (N-N+). The latter oxidizes H202 to evolve 0 2 and regenerate EDTA.
In the presence of I+, a ternary complex of compound I-I+-EDTA is formed, which generates compound
11-I' complex and both nitrogen-centered dication radical (Nf-N+) through intermolecular electron transfer
from EDTA nitrogens. Compound 11-I' complex is further reduced similarly by another molecule of
EDTA to form ferric enzyme, I-, and (Nf-Nf).(Nf-Nf) the oxidation product of EDTA, which may be
released from the active site and, being more reactive, oxidizes H202 to 0 2 at a faster rate to regenerate
EDTA. The existence of (Nf-N+) is suggested from the similarity of its ESR signal with that of single
nitrogen-centered monocation radical (N-N+). EDTA degradation by oxidative decarboxylation due to
two-electron oxidation from the same or bath nitrogen atoms is not evident, and EDTA concentration
remains the same throughout the reactions. While EDTA binds (KD = 15 mM) at or near the iodide
binding site (Bhattacharyya et al. (1993) Biochem. J. 289, 575-580), I+ binds to HRP with a KD value
of 20 & 7 pM. I+ binding in the HRP-CN complex (KD = 20 f 8 pM) indicates that its site is away
from the heme iron center. I+ binding remains unaltered by guaiacol or vice versa, suggesting that I+
binds away from the aromatic donor binding site. As I+ reduction occurs at a saturating concentration of
EDTA, I+ binding at the EDTA site could be excluded. A plot of log KD of I+ binding against various
pHs shows the involvement of an ionizable group on the enzyme having pK, = 4.8, contributed by an
acidic group, deprotonation of which favors I+ binding. Systematic variation of the concentrations of
H202, EDTA, and I+ under steady state condition yields sets of kinetic parameters containing both kinetic
and mechanistic information. Four distinct enzyme species are involved in I+ reduction: native enzyme,
compound I, compound I-I+, and compound 11-I' complex; and rate constants for individual steps are
calculated. The kinetic experiments support the view that an active enzyme-I+-EDTA ternary complex
is formed during I+ reduction by EDTA at the enzyme active site.
 
Date 1995
 
Type Article
PeerReviewed
 
Format application/pdf
 
Identifier http://www.eprints.iicb.res.in/1020/1/biochemistry95.pdf
Adak, Subrata and Bhattacharya, Dipak Kr. and Majumder, Avijit and Bandyopadhyay, Uday and Banerjee, Ranajit K (1995) Concurrent Reduction of Iodine and Oxidation of EDTA at the Active Site of Horseradish Peroxidase: Probing the Iodine Binding Site by Optical Difference Spectroscopy and Steady State Kinetic Analysis for the Formation of Active Enzyme-If -EDTA Ternary Complex for Iodine Reductase Activity. Biochemistry, 34. pp. 12998-13006.
 
Relation http://dx.doi.org/
http://www.eprints.iicb.res.in/1020/