The Origin of the Hydroxyl Radical Oxygen in the Fenton Reaction

doi:10.1016/S0891-5849(96)00432-7

Free Radical Biology and Medicine

Volume 22, Issue 5, 1997, Pages 885-888

https://doi.org/10.1016/S0891-5849(96)00432-7 Get rights and content

Abstract

There is an ongoing discussion in the chemical literature regarding the nature of the highly reactive hydroxyl radical formed from the reaction between ferrous iron and hydrogen peroxide (the Fenton reaction). However, the fundamental experiment of directly determining the source of the hydroxyl radicals formed in the reaction has not yet been carried out. In this study, we have used both hydrogen peroxide and water labeled with ¹⁷O, together with ESR spin trapping, to detect the hydroxyl radicals formed in the reaction. ESR experiments were run in phosphate buffer with 5,5-dimethyl-1-pyrroline N-oxide (DMPO) as a spin trap, and either H₂O₂ or H₂O labeled with ¹⁷O. The hydroxyl radical was generated by addition of Fe²⁺ ion to H₂O₂, or as a control, by photolysis of H₂O₂ in the ESR cavity. Observed ESR spectra were the sum of DMPO/¹⁶OH and DMPO/¹⁷OH radical adduct spectra. Within experimental accuracy, the percentage of ¹⁷O-labeled hydroxyl radical trapped by the DMPO was the same as in the original hydrogen peroxide, for either method of hydroxyl radical generation, indicating that the trapped hydroxyl radical was derived exclusively from hydrogen peroxide and that there was no exchange of oxygen atoms between H₂O₂ and solvent water. Likewise, the complementary reaction with ordinary H₂O₂ and ¹⁷O-labeled water also showed that none of the hydroxyl radical was derived from water. Our results do not preclude the ferryl intermediate, [FeO]²⁺ reacting with DMPO to form DMPO/OH if the ferryl oxygen is derived from H₂O₂ rather than from a water ligand. Copyright © 1997 Elsevier Science Inc.

Introduction

There is an ongoing discussion in the chemical literature regarding the nature of the highly-reactive hydroxyl radical formed from the reaction between ferrous iron and hydrogen peroxide (the Fenton reaction).[1] The classical Fenton mechanism (Eq. (1), or with ligands included, Eq. (1a)) predicts that hydrogen peroxide is reduced at the iron center with generation of free hydroxyl radical. $Fe^{2+} + H_{2} O_{2} → Fe^{3+} + OH^{−} +^{} OH$ $Fe^{II} (H_{2} O)_{6}^{2+} + H_{2} O_{2} → [Fe^{III} (H_{2} O)_{5} OH]^{2+} +^{} OH + H_{2} O$

It has been argued, however, that some, if not all of the hydroxyl radical produced in the Fenton reaction may remain bound at the iron center, either as the [Fe⋯OH]³⁺ or the [FeO]²⁺ intermediate Eq. (2). $Fe^{2+} + H_{2} O_{2} → [FeO]^{2+} + OH^{−}$ These intermediates are proposed to have oxidizing properties similar to, but distinguishable from, free hydroxyl radical, and have been promoted based on comparison of Fenton reaction kinetics with that of the hydroxyl radical generated independently of iron.

Recently Sawyer et al.[2] studied “Fenton reagents” with hydrocarbons as radical scavengers. From product analysis, they concluded that free OH is not the dominant reactant at all, and that with chelated iron, a nucleophilic adduct reacts directly with substrates. Wink et al.[3] used stopped-flow kinetics and competition studies to probe the reaction of a Fenton intermediate with N-nitrosodimethylamine. They proposed a reversible reaction between Fe²⁺ and H₂O₂ to an intermediate X, whose reactivity patterns were consistent with an iron complex and not the OH radical. However, Walling and Amarnath,[4] in a study of the oxidation of a mandelic acid–iron complex by H₂O₂, had previously concluded that both an Fe^IV species and the hydroxyl radical were involved.

For reactions involving free radical intermediates, ESR spectroscopy would appear to be the method of choice, but the OH radical in solution cannot be directly detected by ESR. Thus, indirect techniques must be used, such as spin trapping with 5,5-dimethyl-1-pyrroline-N-oxide (DMPO). Based on relative yields in competitive trapping experiments, Yamazaki and Piette5, 6 presented evidence for the presence of additional oxidizing species other than hydroxyl radical and stated that, although the OH radical is present, it is not all free in solution. However, it has been shown that, under reaction conditions commonly employed, the DMPO/OH adduct can arise as an artifact, such as by oxidation of the DMPO itself followed by reaction with water, or by reaction of a precursor DMPO/superoxide adduct.7, 8

Although the reactivity of iron complexes has been recognized,[2] the existence of “secondary” reactions having different reaction rates for different complexes has not always been considered. Burkitt[7] has shown that such reactions can involve complexed iron with spin trap-hydroxyl radical adducts under commonly employed reaction conditions. For example, the Fe^IIIDTPA (diethylenetriaminepentaacetic acid) complex, in particular, can be reduced by oxidation of the DMPO/OH spin adduct. If not recognized, these reactions can confuse kinetic comparisons and stoichiometric calculations in the Fenton reaction.

Scavenging experiments can also be difficult to interpret if the radicals formed from the scavenger molecules are capable of further reaction. Rush and Koppenol9, 10 studied the Fenton reaction of Fe^IIEDTA, Fe^IIDTPA, and Fe^IIHEDTA with alcohol scavengers and recognized that the nature of the chelator was an important factor. Results with Fe^IIHEDTA suggested an intermediate other than hydroxyl radical, but, in the other two cases, the properties of the intermediate were reported to be similar to the hydroxyl radical. Rahhal and Richter[11] reported that Fe^IIDTPA reacted with H₂O₂ to yield an oxidizing species whose properties in scavenging experiments with tert-butyl alcohol were not consistent with the OH radical Eq. (3). $Fe^{II} (DTPA)^{3−} + H_{2} O_{2} → [Fe^{IV} (DTPA)OH]^{2−} + OH^{−}$ Croft et al.[12] have shown, however, that the radicals formed from alcohols in the Fenton reaction can react with both iron(II) and iron(III) to yield kinetic results that can appear to differ from the hydroxyl radical.

According to existing mechanisms, the oxygen atom in both the ferryl intermediate and the hydroxyl radical originate from hydrogen peroxide, but this has not been tested. Furthermore, if free hydroxyl radical reacts with water, an exchange of oxygen atoms would occur. A more likely possibility is that iron-bound hydroxyl radical species would undergo exchange of oxygen with water. In any case, the fundamental experiment of directly determining the source of the hydroxyl radicals formed has not yet been carried out. In this study, we have used both hydrogen peroxide and water labeled with ¹⁷O, together with ESR spin trapping, to detect the hydroxyl radicals formed in the reaction. We show that, within experimental accuracy, in the Fenton reaction there is no exchange of oxygen atoms between ¹⁷O-labeled H₂O₂ and solvent water or vice-versa.

Section snippets

Materials and Methods

Experiments were run in 100 mM phosphate buffer (pH 7.4) with 100 mM 5,5-dimethyl-1-pyrroline N-oxide (DMPO, Sigma Chemical Co., St. Louis, MO) added as a spin trap. The DMPO was purified twice by vacuum distillation at room temperature and stored at −80°C before use, and the buffer was treated with Chelex® 100 ion-exchange resin (Bio-Rad Laboratories, Hercules, CA) to remove trace heavy metal contaminants. The ¹⁷O-labeled H₂O₂ was from Isotec, Inc. (Miamisburg, OH), and was received as a 2%

Results and Discussion

The observed spectra were the sum of the DMPO/¹⁶OH and DMPO/¹⁷ OH radical adduct spectra. The results of the experiments are given in Table 1. Typical experimental spectra with simulations based on the parameters in Table 1 are shown in Fig. 1.

Within the limits of experimental uncertainty, the percentage of ¹⁷O-labeled hydroxyl radical trapped by the DMPO was the same as in the original hydrogen peroxide for either method of hydroxyl radical generation, indicating that the trapped hydroxyl

References (16)

H.C. Sutton et al.
On the participation of higher oxidation states of iron and copper in Fenton reactions
Free Rad. Biol. Med.
(1989)
I. Yamazaki et al.
ESR Spin-trapping studies on the reaction of Fe²⁺ ions with H₂O₂-reactive species in oxygen toxicity in biology
J. Biol. Chem.
(1990)
J.D. Rush et al.
Oxidizing intermediates in the reaction of ferrous EDTA with hydrogen peroxide
J. Biol. Chem.
(1986)
J.D. Rush et al.
The reaction between ferrous polyaminecarboxylate complexes and hydrogen peroxide: An investigation of the reaction intermediates by stopped flow spectrophotometry
J. Inorg. Biochem.
(1987)
D.R. Duling
Simulation of multiple isotropic spin-trap EPR spectra
J. Magn. Res., Ser. B
(1994)
D.T. Sawyer et al.
Fenton reagents (1:1 Fe^IIL_x/HOOH) react via [L_xFe^IIOOH(BH⁺)] (1) as hydroxylases (RH → ROH), not as generators of free hydroxyl radicals (HO)
J. Am. Chem. Soc.
(1993)
D.A. Wink et al.
The Fenton oxidation mechanism: Reactivities of biologically relevant substrates with two oxidizing intermediates differ from those predicted for the hydroxyl radical
Proc. Natl. Acad. Sci. USA
(1994)
C. Walling et al.
Oxidation of mandelic acid by Fenton's reagent
J. Am. Chem. Soc.
(1982)

There are more references available in the full text version of this article.

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¹: Permanent Address of Roger V. Lloyd: Department of Chemistry, University of Memphis, Memphis, TN 38152.

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Original ContributionThe Origin of the Hydroxyl Radical Oxygen in the Fenton Reaction

Abstract

Introduction

Section snippets

Materials and Methods

Results and Discussion

Free Rad. Biol. Med.

J. Biol. Chem.

J. Biol. Chem.

J. Inorg. Biochem.

J. Magn. Res., Ser. B

Fenton reagents (1:1 FeIILx/HOOH) react via [LxFeIIOOH(BH+)] (1) as hydroxylases (RH → ROH), not as generators of free hydroxyl radicals (HO)

J. Am. Chem. Soc.

The Fenton oxidation mechanism: Reactivities of biologically relevant substrates with two oxidizing intermediates differ from those predicted for the hydroxyl radical

Proc. Natl. Acad. Sci. USA

Oxidation of mandelic acid by Fenton's reagent

J. Am. Chem. Soc.

Original Contribution
The Origin of the Hydroxyl Radical Oxygen in the Fenton Reaction

Fenton reagents (1:1 Fe^IIL_x/HOOH) react via [L_xFe^IIOOH(BH⁺)] (1) as hydroxylases (RH → ROH), not as generators of free hydroxyl radicals (HO)