title: Mechanism of horseradish peroxidase-catalyzed conversion of iodine to iodide in the presence of EDTA and H2O2.
abstract: EDTA not only blocks the horseradish peroxidase (HRP)-catalyzed iodide oxidation to I-3 but also causes an enzymatic conversion of oxidized iodine species to iodide (Banerjee, R. K., De, S. K., Bose, A. K., and Datta, A. G. (1986) J. Biol. Chem. 261, 10592-10597). The EDTA effect on both of these reactions can be withdrawn with a higher concentration of iodide and not with H2O2. Spectral studies indicate a possible interaction of EDTA with HRP as evidenced by the formation of modified compound 1 with H2O2 at 416 nm instead of 412 nm in the absence of EDTA. EDTA causes a hypochromic effect on HRP at 402 nm which undergoes the bathochromic red shift to 416 nm by H2O2. The addition of iodide to the 416 nm complex causes the reappearance of the Soret band of HRP at 402 nm. Among various EDTA analogues tested, N-N-N'-N'-tetramethylethylenediamine (TEMED) is 80% as effective as EDTA in the conversion of I-3 to iodide and produces a spectral shift of HRP similar to EDTA. Interaction of EDTA with HRP is further indicated by the hyperchromic effect of HRP and H2O2 on the absorption of EDTA at 212 nm. The addition of oxidized iodine species produces a new peak at 230 nm due to formation of iodide. EDTA at a higher concentration can effectively displace radioiodide specifically bound to HRP indicating its interaction at the iodide-binding site. The enzyme, after radioiodide displacement with EDTA, shows a characteristic absorption maximum at 416 nm on the addition of H2O2, indicating that EDTA is bound with the enzyme. Both positive and negative circular dichroism spectra of HRP and the HRP.H2O2 complex, characteristic of heme absorption, are altered by EDTA, suggesting an EDTA-induced conformational change at or near the heme region. This is associated with a change of affinity of heme toward H2O2 and azide. It is postulated that EDTA interacts at the iodide-binding site of the HRP inducing a new conformation that blocks iodide oxidation but is suitable to convert iodine to iodide by a redox reaction with H2O2.
authors.affiliation: Department of Physiology, Indian Institute of Chemical Biology, Calcutta.
authors.forename: R K
authors.initials: RK
authors.lastname: Banerjee
mesh_terms: D002384:Catalysis; D004492:Edetic Acid; D005029:Ethylenediamines; D006735:Horseradish Peroxidase; D006861:Hydrogen Peroxide; D007454:Iodides / Q000378:metabolism*; D007455:Iodine; D007700:Kinetics; D010084:Oxidation-Reduction; D010100:Oxygen / Q000378:metabolism; D010544:Peroxidases; D013053:Spectrophotometry
chemical_list: D005029:Ethylenediamines; D007454:Iodides; C005798:N,N,N',N'-tetramethylethylenediamine; D007455:Iodine; D004492:Edetic Acid; D006861:Hydrogen Peroxide; D006735:Horseradish Peroxidase; D010544:Peroxidases; D010100:Oxygen