Ferryl Radical Research Articles

Potential role of ferric hemoglobin in MS pathogenesis: Effects of oxidative stress and extracellular methemoglobin or its degradation products on myelin components

There is a well-documented relationship between cerebral vasculature and multiple sclerosis (MS) lesions: abnormal accumulations of iron have been found in the walls of the dilated veins in cerebral MS plaques. The source of this iron is unknown, but could be related to the recognized phenomenon of capillary and venous hemorrhages leading to blood extravasation. In turn, hemorrhaging leading to hemolysis results in extracellular release of hemoglobin, a reactive molecule that could induce local oxidative stress, inflammation, and tissue damage. Our previous studies with a reduced form of hemoglobin (oxyHb) have demonstrated its ability to cause extensive lipid and protein oxidation in vitro, which would result in membrane destabilization. Here, we investigated in further detail the mechanism by which the more abundant oxidized form of extracellular hemoglobin (metHb), and dissociated hemin, cause direct oxidative damage to myelin components, specifically membrane-mimetic lipid vesicles and myelin basic protein (MBP), a highly-abundant protein in the CNS. Oxidation of lipids was assessed by the formation of conjugated diene/triene and malondialdehyde, and oxidation of MBP was demonstrated by the bityrosine formation and by the change in protein mass. Our results show that metHb causes oxidative damage to MBP and myelin lipids, partly by transferring its hemin moiety to protein and lipid, but mostly as an intact protein possibly via formation of a ferryl radical. These results elucidating the mechanism of extracellular hemoglobin-induced oxidative damage to myelin components support the need for further research into vascular pathology in MS pathogenesis, to gain insight into the role of iron deposits and/or in stimulation of different comorbidities associated with the disease.

Significantly shorter Fe-S bond in cytochrome P450-I is consistent with greater reactivity relative to chloroperoxidase.

Cytochrome P450 (P450) and chloroperoxidase (CPO) are thiolate ligated heme proteins that catalyze the activation of carbon hydrogen bonds. The principal intermediate in these reactions is a ferryl radical species called compound I. P450 compound I (P450-I) is significantly more reactive than CPO-I, which only cleaves activated C-H bonds. To provide insight into the differing reactivities of these intermediates, we examined CPO-I and P450-I with variable temperature Mössbauer and X-ray absorption spectroscopies. These measurements indicate that the Fe-S bond is significantly shorter in P450-I than in CPO-I. This difference in Fe-S bond lengths can be understood in terms of variations in hydrogen bonding patterns within the “cys-pocket” (a portion of the proximal helix that encircles the thiolate ligand). Weaker hydrogen bonding in P450-I results in a shorter Fe-S bond, which enables greater electron donation from the axial-thiolate ligand. This observation may in part explain P450's greater propensity for C-H bond activation.

Monooxygenase, peroxidase and peroxygenase properties and reaction mechanisms of cytochrome P450 enzymes.

This review examines the monooxygenase, peroxidase and peroxygenase properties and reaction mechanisms of cytochrome P450 (CYP) enzymes in bacterial, archaeal and mammalian systems. CYP enzymes catalyze monooxygenation reactions by inserting one oxygen atom from O2 into an enormous number and variety of substrates. The catalytic versatility of CYP stems from its ability to functionalize unactivated carbon-hydrogen (C-H) bonds of substrates through monooxygenation. The oxidative prowess of CYP in catalyzing monooxygenation reactions is attributed primarily to a porphyrin π radical ferryl intermediate known as Compound I (CpdI) (Por•+FeIV=O), or its ferryl radical resonance form (FeIV-O•). CYP-mediated hydroxylations occur via a consensus H atom abstraction/oxygen rebound mechanism involving an initial abstraction by CpdI of a H atom from the substrate, generating a highly-reactive protonated Compound II (CpdII) intermediate (FeIV-OH) and a carbon-centered alkyl radical that rebounds onto the ferryl hydroxyl moiety to yield the hydroxylated substrate. CYP enzymes utilize hydroperoxides, peracids, perborate, percarbonate, periodate, chlorite, iodosobenzene and N-oxides as surrogate oxygen atom donors to oxygenate substrates via the shunt pathway in the absence of NAD(P)H/O2 and reduction-oxidation (redox) auxiliary proteins. It has been difficult to isolate the historically elusive CpdI intermediate in the native NAD(P)H/O2-supported monooxygenase pathway and to determine its precise electronic structure and kinetic and physicochemical properties because of its high reactivity, unstable nature (t½~2 ms) and short life cycle, prompting suggestions for participation in monooxygenation reactions of alternative CYP iron-oxygen intermediates such as the ferric-peroxo anion species (FeIII-OO-), ferric-hydroperoxo species (FeIII-OOH) and FeIII-(H2O2) complex.

Acyl-Carbon Bond Cleaving Cytochrome P450 Enzymes: CYP17A1, CYP19A1 and CYP51A1.

Cytochrome P450 (P450 or CYP) enzymes in their resting state contain the heme-iron in a high-spin FeIII state. Binding of a substrate to a P450 enzyme allows transfer of the first electron, producing a Fe(II) species that reacts with oxygen to generate a low-spin iron superoxide intermediate (FeIII-O-O•) ready to accept the second electron to produce an iron peroxy anion intermediate (a, FeIII-O-O-). In classical monooxygenation reactions, the peroxy anion upon protonation fragments to form the reactive Compound I intermediate (Por•+FeIV=O), or its ferryl radical resonance form (FeIV-O•). However, when the substrate projects a carbonyl functionality, of the type b, at the active site as is the case for reactions catalyzed by CYP17A1, CYP19A1 and CYP51A1, the peroxy anion (FeIII-O-O-) is trapped, yielding a tetrahedral intermediate (c) that fragments to an acyl-carbon cleavage product (d plus an acid). Analogous acyl-carbon cleavage reactions are also catalyzed by certain hepatic P450s and CYP125A1 from Mycobacterium tuberculosis. A further improvisation on the theme is provided by aldehyde deformylases that convert long-chain aliphatic aldehydes to hydrocarbons. CYP17A1 is involved in the biosynthesis of corticoids as well as androgens. The flux toward these two classes of hormones seems to be regulated by cytochrome b 5, at the level of the acyl-carbon cleavage reaction. It is this regulation of CYP17A1 that provides a safety mechanism, ensuring that during corticoid biosynthesis, which requires 17α-hydroxylation by CYP17A1, androgen formation is avoided (Fig. 4.1).

Use of hydrogen peroxide as a biocide: new consideration of its mechanisms of biocidal action

Hydrogen peroxide is extensively used as a biocide, particularly in applications where its decomposition into non-toxic by-products is important. Although increasing information on the biocidal efficacy of hydrogen peroxide is available, there is still little understanding of its biocidal mechanisms of action. This review aims to combine past and novel evidence of interactions between hydrogen peroxide and the microbial cell and its components, while reflecting on alternative applications that make use of gaseous hydrogen peroxide. It is currently believed that the Fenton reaction leading to the production of free hydroxyl radicals is the basis of hydrogen peroxide action and evidence exists for this reaction leading to oxidation of DNA, proteins and membrane lipids in vivo. Investigations of DNA oxidation suggest that the oxidizing radical is the ferryl radical formed from DNA-associated iron, not hydroxyl. Investigations of protein oxidation suggest that selective oxidation of certain proteins might occur, and that vapour-phase hydrogen peroxide is a more potent oxidizer of protein than liquid-phase hydrogen peroxide. Few studies have investigated membrane damage by hydrogen peroxide, though it is suggested that this is important for the biocidal mechanism. No studies have investigated damage to microbial cell components under conditions commonly used for sterilization. Despite extensive studies of hydrogen peroxide toxicity, the mechanism of its action as a biocide requires further investigation.

X-ray absorption spectroscopy of chloroperoxidase compound I: Insight into the reactive intermediate of P450 chemistry

We report the structural characterization of a thiolate-ligated ferryl radical. Using x-ray absorption spectroscopy, we examined chloroperoxidase (CPO) compound I (CPO-I). Our results indicate that CPO-I is an authentic ferryl species with an Fe-O bond of 1.65 A. Axial-ligand interactions result in a remarkably long 2.48-A Fe-S bond. Analogous forms of cytochrome P450 and CPO have been shown to possess virtually identical coordination environments. Thus, it seems likely that our findings provide a good structural description of the elusive P450-I.

PUTTING THE OH WHERE IT BELONGS

ONLY A HANDFUL OF HEME proteins-for example, the medically important family of cytochrome P450 enzymes that metabolize drugs in the body-are capable of attaching hydroxyl groups to inert hydrocarbon substrates. A new X-ray absorption study of a model member of this protein class hints at what allows these enzymes to create reactive iron intermediates that can perform this demanding chemistry without destroying themselves. The active sites of cytochrome P450s contain a heme cofactor whose iron center is coordinated to the protein via a cysteine thiolate. During catalysis, these enzymes are thought to use dioxygen to generate an Fe(IV) = O radical species. This highly reactive ferryl radical species is thought to abstract hydrogen from the hydrocarbon substrate, forming a protonated ferryl that then hydroxylates the substrate. Somehow, this highly reactive ferryl radical species performs hydroxylations without oxidizing the surrounding enzyme. That's a remarkable feat, says Michael T. Green, an assistant ...

Characterization of hydrogen peroxide removal reaction by hemoglobin in the presence of reduced pyridine nucleotides

Hydrogen peroxide removal rates by hemoglobin were enhanced in the presence of reduced pyridine nucleotides. The species which had the activity to oxidize pyridine nucleotides was purified from human blood and identified as hemoglobin A. Hydrogen peroxide removal rates by hemoglobin A without reduced pyridine nucleotides at 0.2 mM hydrogen peroxide were 0.87±0.11 μmol/s/g hemoglobin, and the removal rates using 0.2 mM NADH and NADPH were 2.02±0.20 and 1.96±0.31 μmol/s/g hemoglobin, respectively. We deduced that the removal reaction by hemoglobin included formations of methemoglobin and the ferryl radical and reduction of the latter with pyridine nucleotides. The hydrogen peroxide removal ability by hemoglobin was less than that by catalase but was larger than that by glutathione peroxidase-glutathione reductase system at 0.2 mM hydrogen peroxide. Under acatalasemic conditions, it was suggested that NAD(P)H were important factors to prevent the oxidative degradation of hemoglobin.

Protein radicals in the reaction between H2O2-activated metmyoglobin and bovine serum albumin

Hydrogen peroxide activation of MMb with and without the presence of BSA gave rise to rapid formation of hyper-valent myoglobin species, myoglobin ferryl radical (·MbFe(IV)=O) and/or ferrylmyoglobin (MbFe(IV)=O). Reduction of MbFe(IV)=O showed first-order kinetics for a 1–2 times stoichiometric excess of H2O2 to MMb while a 3–10 times stoichiometric excess of H2O2 resulted in a biphasic reaction pattern. Radical species formed in the reaction between MMb, H2O2 and BSA were influenced by [H2O2] as measured by electron spin resonance (ESR) spectroscopy and resulted in the formation of cross-linking between BSA and myoglobin which was confirmed by SDS-PAGE and subsequent amino acid sequencing. Moreover, dityrosine was formed in the initial phases of the reaction for all concentrations of H2O2. However, initially formed dityrosine was subsequently utilized in reactions employing stoichiometric excess of H2O2 to MMb. The observed breakdown of dityrosine was ascribed to additional radical species formed from the interaction between H2O2 and the hyper-valent iron-center of H2O2-activated MMb.

Peroxidation of linoleate at physiological pH: hemichrome formation by substrate binding protects against metmyoglobin activation by hydrogen peroxide

Peroxidation by metmyoglobin, MbFe(III), by metmyoglobin/hydrogen peroxide, MbFe(III)/H 2O 2 , to yield the myoglobin ferryl radical ( MbFe(IV)=O), or by ferrylmyoglobin, MbFe(IV)=O, was investigated at physiological pH (7.4) in oil-in-water linoleate emulsions. Linoleate peroxidation was followed using second derivative ultraviolet (UV)-spectroscopy for monitoring formation of conjugated dienes and quantitative determination of specific linoleate hydroperoxides by liquid chromatography with photodiode absorption detection. Modifications of myoglobins during lipid peroxidation were followed simultaneously by changes in the Soret absorption band (410 or 424 nm), and in the visible absorption region (from 450 to 700 nm), combined with electron spin resonance (ESR) spectroscopy for direct detection of changes in the spin state of the iron center. In contrast to MbFe(IV)=O, MbFe(III) and MbFe(III)/H 2O 2 were not able to initiate linoleate peroxidation in oil-in-water emulsions, and MbFe(III) was converted, by binding of linoleate (but not methyl linoleate), to a low-spin hemichrome derivate, HMbFe(III), with the distal histidine reversibly bound to the iron center. HMbFe(III) is ineffective in initiating lipid peroxidation and cannot be activated to MbFe(IV)=O or MbFe(IV)=O by addition of moderate amounts of H 2O 2. Addition of MbFe(III) to linoleate emulsions containing H 2O 2 results in the competitive formation of MbFe(IV)=O and HMbFe(III) in favor of HMbFe(III), and little linoleate peroxidation is detected, demonstrating the inherent protection, at physiologic pH, against peroxidation by reversible binding of the substrate to the potential myoglobin catalyst.

Sol−Gel-Encapsulated Heme Proteins. Evidence for CO2 Adducts

Heme proteins have been the subjects of numerous studies of photoinduced ligand dissociation and recombination (e.g., O2, CO, and NO). Much of this work1 is aimed at unraveling the dynamical behavior of myoglobin (Mb), which lacks a crystallographic channel connecting the iron center with the solvent. Despite its importance as a conspicuous metabolic breakdown product, CO2 interactions with metalloproteins are not well understood. CO2 is required for the assembly of the active sites of some multinuclear metalloproteins (e.g., the Ni2 center of urease2). In carbonic anhydrase, zinc-coordinated hydroxide is believed3 to attack CO2, forming a bicarbonate product complex. Deoxyhemoglobin forms carbaminohemoglobin4 in red blood cells upon reaction of CO2 with the amino termini more readily than oxyhemoglobin does; oxygenation in the lungs causes CO2 release. More importantly, there is no direct evidence for the existence of a metal-ligated CO2 adduct of any metalloprotein. In addition to transporting gases, hemoglobin and myoglobin possess enzymatic activity: reactions of the met forms with peroxides generate intermediates5,6 containing ferryl (FeIVdO) and protein radical species that are capable of oxidizing small substrates. In the case of linoleic acid, it has been established7 that substrate oxidation by Mb occurs by direct ferryl oxygen atom transfer. In this paper, we demonstrate that ferrylMb oxidizes carbon monoxide to produce CO2. In addition, we present the first spectroscopic evidence for Fe-CO2 coordination in heme proteins. The reaction7 of metMb with excess hydrogen peroxide generates a metastable intermediate, containing ferryl iron, that decays within minutes to produce degraded forms of myoglobin. However, in a saturated CO solution, Mb is observed8 to produce a gaseous product. Table 1 presents the results of mass spectrometric analyses9 of the gases in sealed pear flasks containing combinations of metMb, H2O2, and CO in phosphate buffer (50 mM, pH 7.0). The m/e ) 44 signal clearly indicates that the product of the catalyzed reaction is CO2. However, the low solubility of CO2 precluded attempts to obtain the electronic spectrum of the product complex under these conditions. Following the suggestion of Ibers10 that liquid CO2 be used in searching for new CO2 complexes, we reasoned that supercritical CO2 would be an even better choice, owing to its gaslike viscosity. Furthermore, supercritical fluids constitute reaction media that are of emerging biotechnological interest.11

Inhibition by activated neutrophils of the Ca2+ pump ATPase of intact red blood cells

Human neutrophils, activated by phorbol myristate acetate in the presence of intact red blood cells (RBCs), caused inhibition of the Ca2+ pump ATPase of the RBCs and fragmentation of the enzyme as well as other membrane proteins. Inhibition of the Ca2+ pump ATPase of intact RBCs was directly related to the neutrophil concentration and the time of incubation. Ca2+ pump ATPase activity was partially protected by the addition of exogenous glutathione-glutathione peroxidase, but not by superoxide dismutase. The addition of sodium azide, a potent inhibitor of endogenous RBC catalase, enhanced inhibition of the Ca2+ pump ATPase of intact RBCs. Examination by SDS-polyacrylamide gel electrophoresis of membrane proteins isolated from RBCs preincubated with activated neutrophils showed gross changes in banding patterns as compared to controls. Thus, a significant amount of methemoglobin appeared to be associated with the membrane proteins, and, in general, protein bands appeared to be more diffuse and less defined than proteins in control lanes. In addition, there was an increase in the low molecular weight protein bands. Using a monoclonal antibody to the Ca2+ pump ATPase, it was shown that the 140 kDa band representing the Ca2+ pump ATPase decreased, with concomitant appearance of two low molecular weight bands running at 8.2 and 6.8 kDa in the membrane proteins from RBCs preincubated with activated neutrophils. The data are interpreted to suggest that inhibition of the Ca2+ pump ATPase in intact RBCs under these conditions occurred as a result of: neutrophil-derived superoxide, dismutation of superoxide, to H2O2, diffusion of H2O2 into RBCs, a Fenton type reaction between oxyhemoglobin, and H2O2 producing hydroxyl radical and/or a ferryl radical capable of promoting protein fragmentation of RBC membrane proteins, including the plasma membrane Ca2+ pump ATPase.

Reaction of Human Hemoglobin HbA0 and Two Cross-Linked Derivatives with Hydrogen Peroxide: Differential Behavior of the Ferryl Intermediate

Functional concerns regarding hemoglobin-based red cell substitutes have generally centered on two parameters: (a) oxygen binding and delivery properties and (b) stabilization of the hemoglobin tetramer to prevent dimerization. Strategic chemical cross-linking and site-directed mutagenesis have produced proteins that have both physiological oxygen binding characteristics and a markedly prolonged retention time in the circulation. The presence of a large amount of redox-active iron outside the red blood cell, however, raises some concerns about the potential for toxic side effects, many involving the production or participation of oxygen free radicals. In the present study, HPLC purified human hemoglobin HbA0 and two derivatives, one cross-linked between the lysine 99 residues of the alpha subunits (α-DBBF) and the other between the lysine 82 residues of the beta subunits (β-DBBF) were tested for their susceptibility to oxidation and oxidative damage caused by H2O2. We show that chemical cross-linking resulting in α-DBBF induces an increased tendency to form ferryl radical in the presence of H2O2 and stabilizes the radical once formed. The in vitro oxidative modification of α-DBBF seen here is a plausible mechanism for some of the in vivo toxicities associated with the infusion of this hemoglobin.

ESR spin-trapping studies of free radicals generated by hydrogen peroxide activation of metmyoglobin

The reaction of metmyoglobin (MetMb) with hydrogen peroxide (H 2 O 2 ) generated a free radical that was detectable by ESR at room temperature only in the presence of the spin trap 5,5-dimethyl-1-pyrroline-N-oxyl (DMPO). The formation of the free radical was favored at pH≥7 and was depressed at low pH. The free radical spin-trapped by DMPO may be a ferryl radical or an amino acid free radical originating from the myoglobin moiety

Toxic DNA damage by hydrogen peroxide through the Fenton reaction in vivo and in vitro.

Exposure of Escherichia coli to low concentrations of hydrogen peroxide results in DNA damage that causes mutagenesis and kills the bacteria, whereas higher concentrations of peroxide reduce the amount of such damage. Earlier studies indicated that the direct DNA oxidant is a derivative of hydrogen peroxide whose formation is dependent on cell metabolism. The generation of this oxidant depends on the availability of both reducing equivalents and an iron species, which together mediate a Fenton reaction in which ferrous iron reduces hydrogen peroxide to a reactive radical. An in vitro Fenton system was established that generates DNA strand breaks and inactivates bacteriophage and that also reproduces the suppression of DNA damage by high concentrations of peroxide. The direct DNA oxidant both in vivo and in this in vitro system exhibits reactivity unlike that of a free hydroxyl radical and may instead be a ferryl radical.