Reaction of haem containing proteins and enzymes with hydroperoxides: the radical view

The reaction between hydroperoxides and the haem group of proteins and enzymes is important for the function of many enzymes but has also been implicated in a number of pathological conditions where oxygen binding proteins interact with hydrogen peroxide or other peroxides. The haem group in the oxidized Fe3+ (ferric) state reacts with hydroperoxides with a formation of the Fe4+=O (oxoferryl) haem state and a free radical primarily located on the pi-system of the haem. The radical is then transferred to an amino acid residue of the protein and undergoes further transfer and transformation processes. The free radicals formed in this reaction are reviewed for a number of proteins and enzymes. Their previously published EPR spectra are analysed in a comparative way. The radicals directly detected in most systems are tyrosyl radicals and the peroxyl radicals formed on tryptophan and possibly cysteine. The locations of the radicals in the proteins have been reported as follows: Tyr133 in soybean leghaemoglobin; alphaTyr42, alphaTrp14, betaTrp15, betaCys93, (alphaTyr24-alphaHis20), all in the alpha- and beta-subunits of human haemoglobin; Tyr103, Tyr151 and Trp14 in sperm whale myoglobin; Tyr103, Tyr146 and Trp14 in horse myoglobin; Trp14, Tyr103 and Cys110 in human Mb. The sequence of events leading to radical formation, transformation and transfer, both intra- and intermolecularly, is considered. The free radicals induced by peroxides in the enzymes are reviewed. Those include: lignin peroxidase, cytochrome c peroxidase, cytochrome c oxidase, turnip isoperoxidase 7, bovine catalase, two isoforms of prostaglandin H synthase, Mycobacterium tuberculosis and Synechocystis PCC6803 catalase-peroxidases.     

Maturation of the cytochrome cd1 nitrite reductase NirS from Pseudomonas aeruginosa requires transient interactions between the three proteins NirS,NirN and NirF

The periplasmic cytochrome cd₁ nitrite reductase NirS occurring in denitrifying bacteria such as the human pathogen Pseudomonas aeruginosa contains the essential tetrapyrrole cofactors haem c and haem d₁. Whereas the haem c is incorporated into NirS by the cytochrome c maturation system I, nothing is known about the insertion of the haem d₁ into NirS. Here, we show by co-immunoprecipitation that NirS interacts with the potential haem d₁ insertion protein NirN in vivo. This NirS–NirN interaction is dependent on the presence of the putative haem d₁ biosynthesis enzyme NirF. Further, we show by affinity co-purification that NirS also directly interacts with NirF. Additionally, NirF is shown to be a membrane anchored lipoprotein in P. aeruginosa. Finally, the analysis by UV–visible absorption spectroscopy of the periplasmic protein fractions prepared from the P. aeruginosa WT (wild-type) and a P. aeruginosa ΔnirN mutant shows that the cofactor content of NirS is altered in the absence of NirN. Based on our results, we propose a potential model for the maturation of NirS in which the three proteins NirS, NirN and NirF form a transient, membrane-associated complex in order to achieve the last step of haem d₁ biosynthesis and insertion of the cofactor into NirS.     

Characterisation of Desulfovibrio vulgaris haem b synthase,a radical SAM family member

An alternative route for haem b biosynthesis is operative in sulfate-reducing bacteria of the Desulfovibrio genus and in methanogenic Archaea. This pathway diverges from the canonical one at the level of uroporphyrinogen III and progresses via a distinct branch, where sirohaem acts as an intermediate precursor being converted into haem b by a set of novel enzymes, named the alternative haem biosynthetic proteins (Ahb). In this work, we report the biochemical characterisation of the Desulfovibrio vulgaris AhbD enzyme that catalyses the last step of the pathway. Mass spectrometry analysis showed that AhbD promotes the cleavage of S-adenosylmethionine (SAM) and converts iron-coproporphyrin III via two oxidative decarboxylations to yield haem b, methionine and the 5′-deoxyadenosyl radical. Electron paramagnetic resonance spectroscopy studies demonstrated that AhbD contains two [4Fe–4S]^2 +/1 + centres and that binding of the substrates S-adenosylmethionine and iron-coproporphyrin III induces conformational modifications in both centres. Amino acid sequence comparisons indicated that D. vulgaris AhbD belongs to the radical SAM protein superfamily, with a GGE-like motif and two cysteine-rich sequences typical for ligation of SAM molecules and iron-sulfur clusters, respectively. A structural model of D. vulgaris AhbD with putative binding pockets for the iron-sulfur centres and the substrates SAM and iron-coproporphyrin III is discussed.     

Modulation of nucleotide binding to the catalytic sites of thermophilic F1-ATPase by the ε subunit: Implication for the role of the ε subunit in ATP synthesis

Effect of ε subunit on the nucleotide binding to the catalytic sites of F₁-ATPase from the thermophilic Bacillus PS3 (TF₁) has been tested by using α₃β₃γ and α₃β₃γε complexes of TF₁ containing βTyr341 to Trp substitution. The nucleotide binding was assessed with fluorescence quenching of the introduced Trp. The presence of the ε subunit weakened ADP binding to each catalytic site, especially to the highest affinity site. This effect was also observed when GDP or IDP was used. The ratio of the affinity of the lowest to the highest nucleotide binding sites had changed two orders of magnitude by the ε subunit. The differences may relate to the energy required for the binding change in the ATP synthesis reaction and contribute to the efficient ATP synthesis.     

A Hydrogen-Donating Monohydroxamate Scavenges Ferryl Myoglobin Radicals

The addition of 25μM hydrogen peroxide to 20μM metmyoglobin produces ferryl (Fe^IV = O) myoglobin. Optical spectroscopy shows that the ferryl species reaches a maximum concentration (60-70% of total haem) after 10 minutes and decays slowly (hours). Low temperature EPR spectroscopy of the high spin metmyoglobin (g = 6) signal is consistent with these findings. At this low peroxide concentration there is no evidence for iron release from the haem. At least two free radicals are detectable by EPR immediately after H₂O₂ addition, but decay completely after ten minutes. However, a longer-lived radical is observed at lower concentrations that is still present after 90 minutes. The monohydroxamate N-methylbutyro-hydroxamic acid (NMBH) increases the rate of decay of the fenyl species. In the presence of NMBH, none of the protein-bound free radicals are detectable; instead nitroxide radicals produced by oxidation of the hydroxamate group are observed. Similar results are observed with the trihydroxamate, desferoxamine. “Ferryl myoglobin” is still able to initiate lipid peroxidation even after the short-lived protein free radicals are no longer detectable (E.S.R. Newman, C.A. Rice-Evans and M.J. Davies (1991) Biochemical and Biophysical Research Communications 179, 1414-1419). It is suggested that the longer-lived protein radicals described here may be partly responsible for this effect. The mechanism of inhibition of initiation of lipid peroxidation by hydroxamate drugs, such as NMBH, may therefore be due to reduction of the protein-derived radicals, rather than reduction of ferryl haem.     

Recent progress on obtaining theoretical and experimental support for the “E-pathway hypothesis” of coupled transmembrane electron and proton transfer in dihaem-containing quinol:fumarate reductase

Reconciliation of apparently contradictory experimental results obtained on the quinol: fumarate reductase (QFR), a dihaem-containing respiratory membrane protein complex from Wolinella succinogenes, was previously obtained by the proposal of the so-called E-pathway hypothesis. According to this hypothesis, transmembrane electron transfer via the haem groups is strictly coupled to co-transfer of protons via a transiently established, novel pathway, proposed to contain the side chain of residue Glu-C180 and the distal haem ring-C propionate as the most prominent components. This hypothesis has recently been supported by both theoretical and experimental results. Multiconformation continuum electrostatics calculations predict Glu-C180 to undergo a combination of proton uptake and conformational change upon haem reduction. Strong experimental support for the proposed role of Glu-C180 in the context of the “E-pathway hypothesis” is provided by the effects of replacing Glu-C180 with Gln or Ile by site-directed mutagenesis, the consequences of these mutations for the viability of the resulting mutants, together with the structural and functional characterisation of the corresponding variant enzymes, and the comparison of redox-induced Fourier-transform infrared (FTIR) difference spectra for the wild type and Glu-C180 → Gln variant. A possible haem propionate involvement has recently been supported by combining ¹³C-haem propionate labelling with redox-induced FTIR difference spectroscopy.     

Myoglobin-H2O2 catalyzes the oxidation of β-ketoacids to α-dicarbonyls: mechanism and implications in ketosis

Acetoacetate (AA) and 2-methylacetoacetate (MAA) are accumulated in metabolic disorders such as diabetes and isoleucinemia. Here we examine the mechanism of AA and MAA aerobic oxidation initiated by myoglobin (Mb)/H₂O₂. We propose a chemiluminescent route involving a dioxetanone intermediate whose thermolysis yields triplet α-dicarbonyl species (methylglyoxal and diacetyl). The observed ultraweak chemiluminescence increased linearly on raising the concentration of either Mb (10-500 μM) or AA (10-100 mM). Oxygen uptake studies revealed that MAA is almost a 100-fold more reactive than AA. EPR spin-trapping studies with MNP/MAA revealed the intermediacy of an α-carbon-centered radical and acetyl radical. The latter radical, probably derived from triplet diacetyl, is totally suppressed by sorbate, a well-known quencher of triplet carbonyls. Furthermore, an EPR signal assignable to MNP-AA^• adduct was observed and confirmed by isotope effects. Oxygen consumption and α-dicarbonyl yield were shown to be dependent on AA or MAA concentrations (1-50 mM) and on H₂O₂ or tert-butOOH added to the Mb-containing reaction mixtures. That ferrylMb is involved in a peroxidase cycle acting on the substrates is suggested by the reaction pH profiles and immunospin-trapping experiments. The generation of radicals and triplet dicarbonyl products by Mb/H₂O₂/β-ketoacids may contribute to the adverse health effects of ketogenic unbalance.     

Enhanced catalytic site thermal stability of cold-adapted esterase EstK by a W208Y mutation

Hydrophobic interactions are known to play an important role for cold-adaptation of proteins; however, the role of amino acid residue, Trp, has not been systematically investigated. The extracellular esterase, EstK, which was isolated from the cold-adapted bacterium Pseudomonas mandelii, has 5 Trp residues. In this study, the effects of Trp mutation on thermal stability, catalytic activity, and conformational change of EstK were investigated. Among the 5 Trp residues, W²⁰⁸ was the most crucial in maintaining structural conformation and thermal stability of the enzyme. Surprisingly, mutation of W²⁰⁸ to Tyr (W²⁰⁸Y) showed an increased catalytic site thermal stability at ambient temperatures with a 13-fold increase in the activity at 40 °C compared to wild-type EstK. The structure model of W²⁰⁸Y suggested that Y²⁰⁸ could form a hydrogen bond with D³⁰⁸, which is located next to catalytic residue H³⁰⁷, stabilizing the catalytic domain. Interestingly, Tyr was conserved in the corresponding position of hyper-thermophilic esterases EstE1 and AFEST, which are active at high temperatures. Our study provides a novel insight into the engineering of the catalytic site of cold-adapted enzymes with increased thermal stability and catalytic activity at ambient temperatures.     

An NMR-based docking model for the physiological transient complex between cytochrome f and cytochrome c6

The physiological transient complex between cytochrome f (Cf) and cytochrome c₆ (Cc₆) from the cyanobacterium Nostoc sp. PCC 7119 has been analysed by NMR spectroscopy. The binding constant at low ionic strength is 8 ± 2 mM⁻¹, and the binding site of Cc₆ for Cf is localized around its exposed haem edge. On the basis of the experimental data, the resulting docking simulations suggest that Cc₆ binds to Cf in a fashion that is analogous to that of plastocyanin but differs between prokaryotes and eukaryotes.     

Structural comparisons of arachidonic acid-induced radicals formed by prostaglandin H synthase-1 and -2

Cyclooxygenase catalysis by prostaglandin H synthase (PGHS)-1 and -2 involves reaction of a peroxide-induced Tyr385 radical with arachidonic acid (AA) to form an AA radical that reacts with O₂. The potential for isomeric AA radicals and formation of an alternate tyrosyl radical at Tyr504 complicate analysis of radical intermediates. We compared the EPR spectra of PGHS-1 and -2 reacted with peroxide and AA or specifically deuterated AA in anaerobic, single-turnover experiments. With peroxide-treated PGHS-2, the carbon-centered radical observed after AA addition was consistently a pentadienyl radical; a variable wide-singlet (WS) contribution from mixture of Tyr385 and Tyr504 radicals was also present. Analogous reactions with PGHS-1 produced EPR signals consistent with varying proportions of pentadienyl and tyrosyl radicals, and two additional EPR signals. One, insensitive to oxygen exposure, is the narrow singlet tyrosyl radical with clear hyperfine features found previously in inhibitor-pretreated PGHS-1. The second type of EPR signal is a narrow singlet lacking detailed hyperfine features that disappeared upon oxygen exposure. This signal was previously ascribed to an allyl radical, but high field EPR analysis indicated that ~ 90% of the signal originates from a novel tyrosyl radical, with a small contribution from a carbon-centered species. The radical kinetics could be resolved by global analysis of EPR spectra of samples trapped at various times during anaerobic reaction of PGHS-1 with a mixture of peroxide and AA. The improved understanding of the dynamics of AA and tyrosyl radicals in PGHS-1 and -2 will be useful for elucidating details of the cyclooxygenase mechanism, particularly the H-transfer between tyrosyl radical and AA.     

Oxidizing substrate specificity of Mycobacterium tuberculosis alkyl hydroperoxide reductase E: kinetics and mechanisms of oxidation and overoxidation

Alkyl hydroperoxide reductase E (AhpE), a novel subgroup of the peroxiredoxin family, comprises Mycobacterium tuberculosis AhpE (MtAhpE) and AhpE-like proteins present in many bacteria and archaea, for which functional characterization is scarce. We previously reported that MtAhpE reacted ~ 10³ times faster with peroxynitrite than with hydrogen peroxide, but the molecular reasons for that remained unknown. Herein, we investigated the oxidizing substrate specificity and the oxidative inactivation of the enzyme. In most cases, both peroxidatic thiol oxidation and sulfenic acid overoxidation followed a trend in which those peroxides with the lower leaving-group pK_a reacted faster than others. These data are in agreement with the accepted mechanisms of thiol oxidation and support that overoxidation occurs through sulfenate anion reaction with the protonated peroxide. However, MtAhpE oxidation and overoxidation by fatty acid-derived hydroperoxides (~ 10⁸ and 10⁵ M^− 1 s^− 1, respectively, at pH 7.4 and 25 °C) were much faster than expected according to the Brønsted relationship with leaving-group pK_a. A stoichiometric reduction of the arachidonic acid hydroperoxide 15-HpETE to its corresponding alcohol was confirmed. Interactions of fatty acid hydroperoxides with a hydrophobic groove present on the reduced MtAhpE surface could be the basis of their surprisingly fast reactivity.     

Formation of methyl radicals derived from cumene hydroperoxide in reconstructed human epidermis: an EPR spin trapping confirmation by using 13C-substitution

Dermal exposure to cumene hydroperoxide (CumOOH) during manufacturing processes is a toxicological issue for the industry. Its genotoxicity, mutagenic action, ability to promote skin tumour, capacity to induce epidermal hyperplasia, and aptitude to induce allergic and irritant skin contact dermatitis are well known. These toxic effects appear to be mediated through the activation to free radical species such as hydroxyl, alkoxyl, and alkyl radicals characterised basically by electron paramagnetic resonance (EPR) and spin-trapping (ST) techniques. To be a skin sensitiser CumOOH needs to covalently bind to skin proteins in the epidermis to form the antigenic entity triggering the immunotoxic reaction. Cleavage of the O–O bond allows formation of unstable CumO^?/CumOO^? radicals rearranging to longer half-life specific carbon-centred radicals R^? proposed to be at the origin of the antigen formation. Nevertheless, it is not still clear which R^? is precisely formed in the epidermis and thus involved in the sensitisation process. The aim of this work was to elucidate in conditions closer to real-life sensitisation which specific R^? are formed in a 3D reconstructed human epidermis (RHE) model by using ¹³C-substituted CumOOH at carbon positions precursors of potentially reactive radicals and EPR-ST. We demonstrated that most probably methyl radicals derived from β-scission of CumO^? radicals occur in RHE through a one-electron reductive pathway suggesting that these could be involved in the antigen formation inducing skin sensitisation. We also describe a coupling between nitroxide radicals and β position ¹³C atoms that could be of an added value to the very few examples existing for the coupling of radicals with ¹³C atoms.     

Functional properties of type I and type II cytochromes c3 from Desulfovibrio africanus

Type I cytochrome c₃ is a key protein in the bioenergetic metabolism of Desulfovibrio spp., mediating electron transfer between periplasmic hydrogenase and multihaem cytochromes associated with membrane bound complexes, such as type II cytochrome c₃. This work presents the NMR assignment of the haem substituents in type I cytochrome c₃ isolated from Desulfovibrio africanus and the thermodynamic and kinetic characterisation of type I and type II cytochromes c₃ belonging to the same organism. It is shown that the redox properties of the two proteins allow electrons to be transferred between them in the physiologically relevant direction with the release of energised protons close to the membrane where they can be used by the ATP synthase.     

Sensitivity of oocyte-expressed epithelial Na channel to glibenclamide

The effect of glibenclamide on heterologously expressed amiloride-sensitive sodium channels (ENaCs) was investigated in Xenopus oocytes. The ENaC is a heteromer and consists of α-, β- and γ-subunits and the α- and β-subunits have previously been shown to confer sensitivity to glibenclamide. We coexpressed either colonic rat α- (rα) or guinea-pig α-subunit (gpα) with Xenopus βγ-subunits. The gpαxβγ was significantly stimulated by glibenclamide (100 μM) (184±15%), whereas the rα-combination was slightly down-regulated by the sulfonylurea (79±4%). The stimulating effect did not interfere with Na⁺-self-inhibition resulting from intracellular accumulation of Na⁺-ions. We exchanged cytosolic termini between both orthologs but the gpα-chimera with the termini from rat retained sensitivity to glibenclamide. The effect of glibenclamide on Xenopus ENaC (xENaC) was inhibited by ADP-β-S but not by ATP-γ-S, when applied intracellularly. Intracellular loading with Na⁺-ions after inhibition of Na⁺/K⁺-ATPases with ouabain prevented an up-regulation of ENaC activity by glibenclamide. Pretreatment of oocytes expressing xENaC with edelfosine (ET-18-OCH₃) slightly reduced stimulation of I_ami (118±12%; control: 132±9%) while phosphatidylinositol-4,5-biphosphate (PIP₂) significantly reduced the effect of glibenclamide to 101±3%.     

Extreme temperature tolerance of a hyperthermophilic protein coupled to residual structure in the unfolded state

Understanding the mechanisms that dictate protein stability is of large relevance, for instance, to enable design of temperature-tolerant enzymes with high enzymatic activity over a broad temperature interval. In an effort to identify such mechanisms, we have performed a detailed comparative study of the folding thermodynamics and kinetics of the ribosomal protein S16 isolated from a mesophilic (S16_meso) and hyperthermophilic (S16_thermo) bacterium by using a variety of biophysical methods. As basis for the study, the 2.0 Å X-ray structure of S16_thermo was solved using single wavelength anomalous dispersion phasing. Thermal unfolding experiments yielded midpoints of 59 and 111 °C with associated changes in heat capacity upon unfolding (ΔC_p⁰) of 6.4 and 3.3 kJ mol^− 1 K^− 1, respectively. A strong linear correlation between ΔC_p⁰ and melting temperature (T_m) was observed for the wild-type proteins and mutated variants, suggesting that these variables are intimately connected. Stopped-flow fluorescence spectroscopy shows that S16_meso folds through an apparent two-state model, whereas S16_thermo folds through a more complex mechanism with a marked curvature in the refolding limb indicating the presence of a folding intermediate. Time-resolved energy transfer between Trp and N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-yl)methyl iodoacetamide of proteins mutated at selected positions shows that the denatured state ensemble of S16_thermo is more compact relative to S16_meso. Taken together, our results suggest the presence of residual structure in the denatured state ensemble of S16_thermo that appears to account for the large difference in quantified ΔC_p⁰ values and, in turn, parts of the observed extreme thermal stability of S16_thermo. These observations may be of general importance in the design of robust enzymes that are highly active over a wide temperature span.     

Nitric oxide, cytochrome c oxidase and myoglobin: competition and reaction pathways

It is relevant to cell physiology that nitric oxide (NO) reacts with both cytochrome oxidase (CcOX) and oxygenated myoglobin (MbO(2)). In this respect, it has been proposed [Pearce, L.L., et al. (2002) J. Biol. Chem. 277, 13556-13562] that (i) CcOX in turnover out-competes MbO(2) for NO, and (ii) NO bound to reduced CcOX is "metabolized" in the active site to nitrite by reacting with O(2). In contrast, rapid kinetics experiments reported in this study show that (i) upon mixing NO with MbO(2) and CcOX in turnover, MbO(2) out-competes the oxidase for NO and (ii) after mixing nitrosylated CcOX with O(2) in the presence of MbO(2), NO (and not nitrite) dissociates from the enzyme causing myoglobin oxidation.     

A conserved haem redox and trafficking pathway for cofactor attachment

A pathway for cytochrome c maturation (Ccm) in bacteria, archaea and eukaryotes (mitochondria) requires the genes encoding eight membrane proteins (CcmABCDEFGH). The CcmABCDE proteins are proposed to traffic haem to the cytochrome c synthetase (CcmF/H) for covalent attachment to cytochrome c by unknown mechanisms. For the first time, we purify pathway complexes with trapped haem to elucidate the molecular mechanisms of haem binding, trafficking and redox control. We discovered an early step in trafficking that involves oxidation of haem (to Fe³⁺), yet the final attachment requires reduced haem (Fe²⁺). Surprisingly, CcmF is a cytochrome b with a haem never before realized, and in vitro, CcmF functions as a quinol:haem oxidoreductase. Thus, this ancient pathway has conserved and orchestrated mechanisms for trafficking, storing and reducing haem, which assure its use for cytochrome c synthesis even in limiting haem (iron) environments and reducing haem in oxidizing environments.     

Direct detection of radical generation in rat liver nuclei on treatment with tumour-promoting hydroperoxides and related compounds

EPR spin trapping has been employed to directly detect radical production in isolated rat nuclei on exposure to a variety of hydroperoxides and related compounds which are known, or suspect, tumour promoters. The hydroperoxides, in the absence of reducing equivalents, undergo oxidative cleavage, generating peroxyl radicals. In the presence of NADPH (and to a lesser extent NADH) reductive cleavage of the OO bond generates alkoxyl radicals. These radicals undergo subsequent rearrangements and reactions (dependent on the structure of the alkoxyl radical), generating carbon-centred radicals. Acyl peroxides and peracids appear to undergo only reductive cleavage of the OO bond. With peracids this cleavage can generate aryl carboxyl (RCO₂·) or hydroxyl radicals (HO·); with acyl peroxides, aryl carboxyl radicals are formed and, in the case of t-butyl peroxybenzoate, alkoxyl radicals (RO·). The radicals detected with each peroxide are similar in type to those detected in the rat liver microsomal fraction, although the extent of radical production is lower. The subsequent reactions of the initially generated radicals are similar to those determined in homogenous chemical systems, suggesting that they are in free solution. Experiments with NADPH/NADH, heat denaturation of the nuclei and various inhibitors suggest that radical generation is an enzymatic process catalysed by haemproteins, in particular cytochrome P-450, and that NADPH/cytochrome P-450 reductase is involved in the reductive cleavage of the OO bond. The generation of these radicals by the rat liver nuclear fraction is potentially highly damaging for the cell due to the proximity of the generating source to DNA. Several previous studies have shown that some of the radicals detected in this study, such as aryl carboxyl and aryl radicals, can damage DNA, via various reactions which results in the generation of strand breaks and adducts to DNA bases: these processes are suggested to play an important role in the tumour promoting activity of these hydroperoxides and related compounds.     

Formation of Hydroxyl Radicals in Biological Systems. Does Myoglobin Stimulate Hydroxyl Radical Formation from Hydrogen Peroxide?

Incubation of horse-heart oxymyoglobin or metmyoglobin with excess H₂O₂ causes formation of myoglobin(IV), followed by haem degradation. At the time when haem degradation is observed, hydroxyl radicals (.OH) can be detected in the reaction mixture by their ability to degrade the sugar deoxyribose. Detection of hydroxyl radicals can be decreased by transferrin or by OH scavengers (mannitol, arginine, phenylalanine) but not by urea. Neither transferrin nor any of these scavengers inhibit the haem degradation. It is concluded that intact oxymyoglobin or metmyoglobin molecules do not react with H₂O₂ to form OH detectable by deoxyribose, but that H₂O₂ eventually leads to release of iron ions from the proteins. These released iron ions can react to form OH outside the protein or close to its surface. Salicylate and the iron chelator desferrioxamine stabilize myoglobin and prevent haem degradation. The biological importance of OH generated using iron ions released from myoglobin by H₂O₂ is discussed in relation to myocardial reoxygenation injury.     

Removal of H₂O₂ and generation of superoxide radical: role of cytochrome c and NADH

In cells, mitochondria, endoplasmic reticulum, and peroxisomes are the major sources of reactive oxygen species (ROS) under physiological and pathophysiological conditions. Cytochrome c (cyt c) is known to participate in mitochondrial electron transport and has antioxidant and peroxidase activities. Under oxidative or nitrative stress, the peroxidase activity of Fe³⁺cyt c is increased. The level of NADH is also increased under pathophysiological conditions such as ischemia and diabetes and a concurrent increase in hydrogen peroxide (H₂O₂) production occurs. Studies were performed to understand the related mechanisms of radical generation and NADH oxidation by Fe³⁺cyt c in the presence of H₂O₂. Electron paramagnetic resonance (EPR) spin trapping studies using 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) were performed with NADH, Fe³⁺cyt c, and H₂O₂ in the presence of methyl-β-cyclodextrin. An EPR spectrum corresponding to the superoxide radical adduct of DMPO encapsulated in methyl-β-cyclodextrin was obtained. This EPR signal was quenched by the addition of the superoxide scavenging enzyme Cu,Zn-superoxide dismutase (SOD1). The amount of superoxide radical adduct formed from the oxidation of NADH by the peroxidase activity of Fe³⁺cyt c increased with NADH and H₂O₂ concentration. From these results, we propose a mechanism in which the peroxidase activity of Fe³⁺cyt c oxidizes NADH to NAD^•, which in turn donates an electron to O₂, resulting in superoxide radical formation. A UV-visible spectroscopic study shows that Fe³⁺cyt c is reduced in the presence of both NADH and H₂O₂. Our results suggest that Fe³⁺cyt c could have a novel role in the deleterious effects of ischemia/reperfusion and diabetes due to increased production of superoxide radical. In addition, Fe³⁺cyt c may play a key role in the mitochondrial “ROS-induced ROS-release” signaling and in mitochondrial and cellular injury/death. The increased oxidation of NADH and generation of superoxide radical by this mechanism may have implications for the regulation of apoptotic cell death, endothelial dysfunction, and neurological diseases. We also propose an alternative electron transfer pathway, which may protect mitochondria and mitochondrial proteins from oxidative damage.