From chlorite dismutase towards HemQ - the role of the proximal H-bonding network in haeme binding.

Chlorite dismutase (Cld) and HemQ are structurally and phylogenetically closely related haeme enzymes differing fundamentally in their enzymatic properties. Clds are able to convert chlorite into chloride and dioxygen, whereas HemQ is proposed to be involved in the haeme b synthesis of Gram-positive bacteria. A striking difference between these protein families concerns the proximal haeme cavity architecture. The pronounced H-bonding network in Cld, which includes the proximal ligand histidine and fully conserved glutamate and lysine residues, is missing in HemQ. In order to understand the functional consequences of this clearly evident difference, specific hydrogen bonds in Cld from 'Candidatus Nitrospira defluvii' (NdCld) were disrupted by mutagenesis. The resulting variants (E210A and K141E) were analysed by a broad set of spectroscopic (UV-vis, EPR and resonance Raman), calorimetric and kinetic methods. It is demonstrated that the haeme cavity architecture in these protein families is very susceptible to modification at the proximal site. The observed consequences of such structural variations include a significant decrease in thermal stability and also affinity between haeme b and the protein, a partial collapse of the distal cavity accompanied by an increased percentage of low-spin state for the E210A variant, lowered enzymatic activity concomitant with higher susceptibility to self-inactivation. The high-spin (HS) ligand fluoride is shown to exhibit a stabilizing effect and partially restore wild-type Cld structure and function. The data are discussed with respect to known structure-function relationships of Clds and the proposed function of HemQ as a coprohaeme decarboxylase in the last step of haeme biosynthesis in Firmicutes and Actinobacteria.

Fermented and extruded wheat bran in piglet diets: impact on performance, intestinal morphology, microbial metabolites in chyme and blood lipid radicals.

The aim of the present study was to evaluate the influence of native, fermented and extruded wheat bran on the performance and intestinal morphology of piglets. Additionally, short-chain fatty acids (SCFA), biogenic amines, ammonia, lactic acid, pH as well as E. coli and lactic acid bacterial counts were analysed in digesta samples from three gut sections. Furthermore, the antioxidant potential in blood samples was evaluated based on the lipid radicals formed. For this purpose, 48 newly weaned piglets (28 d old) were allocated to one of the four different dietary treatment groups: no wheat bran (Control), native wheat bran, fermented wheat bran as well as extruded wheat bran. Wheat bran variants were included at 150 g/kg into the diets. All diets were mixed to reach the calculated isonitrogenic nutrient contents. Gut tissue and digesta samples were collected from the proximal jejunum, the terminal ileum and the colon ascendens, blood samples directly at slaughter. Although none of the dietary interventions had an impact on performance parameters, the amount of goblet cells in the ileum was increased upon feeding native and extruded wheat bran, compared to fermented bran (p < 0.05). The E. coli counts in colonic chyme were significantly lower (p < 0.05) in the Control group compared to the groups fed with wheat bran. The concentration of SCFA showed differences for minor compounds (p < 0.05), while linear contrast analyses revealed a reduced concentration of total SCFA in the colon following the feeding of modified wheat bran compared to native wheat bran. This may suggest that several compounds are more easily digested already in the ileum, resulting in a reduced nutrient flow into the large intestine and therefore less unexploited digesta is available as substrate for the microorganisms there. Fermentation also resulted in a significant decrease of methylamine in the colon (p < 0.05), while other biogenic amines in the ileum and colon showed no statistically significant differences. The formation of lipid radicals was decreased (p < 0.05) after feeding native wheat bran compared to the Control group. These results suggest that fermentation and extrusion of wheat bran exert some different impact regarding their physiological mode of action.

Eukaryotic Catalase-Peroxidase: The Role of the Trp-Tyr-Met Adduct in Protein Stability, Substrate Accessibility, and Catalysis of Hydrogen Peroxide Dismutation.

Recently, it was demonstrated that bifunctional catalase-peroxidases (KatGs) are found not only in archaea and bacteria but also in lower eukaryotes. Structural studies and preliminary biochemical data of the secreted KatG from the rice pathogen Magnaporthe grisea (MagKatG2) suggested both similar and novel features when compared to those of the prokaryotic counterparts studied so far. In this work, we demonstrate the role of the autocatalytically formed redox-active Trp140-Tyr273-Met299 adduct of MagKatG2 in (i) the maintenance of the active site architecture, (ii) the catalysis of hydrogen peroxide dismutation, and (iii) the protein stability by comparing wild-type MagKatG2 with the single mutants Trp140Phe, Tyr273Phe, and Met299Ala. The impact of disruption of the covalent bonds between the adduct residues on the spectral signatures and heme cavity architecture was small. By contrast, loss of its integrity converts bifunctional MagKatG2 to a monofunctional peroxidase of significantly reduced thermal stability. It increases the accessibility of ligands due to the increased flexibility of the KatG-typical large loop 1 (LL1), which contributes to the substrate access channel and anchors at the adduct Tyr. We discuss these data with respect to those known from prokaryotic KatGs and in addition present a high-resolution structure of an oxoiron compound of MagKatG2.

Probing the Solution Structure of IκB Kinase (IKK) Subunit γ and Its Interaction with Kaposi Sarcoma-associated Herpes Virus Flice-interacting Protein and IKK Subunit ß by EPR Spectroscopy.

Viral flice-interacting protein (vFLIP), encoded by the oncogenic Kaposi sarcoma-associated herpes virus (KSHV), constitutively activates the canonical nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) pathway. This is achieved through subversion of the IκB kinase (IKK) complex (or signalosome), which involves a physical interaction between vFLIP and the modulatory subunit IKKγ. Although this interaction has been examined both in vivo and in vitro, the mechanism by which vFLIP activates the kinase remains to be determined. Because IKKγ functions as a scaffold, recruiting both vFLIP and the IKKα/ß subunits, it has been proposed that binding of vFLIP could trigger a structural rearrangement in IKKγ conducive to activation. To investigate this hypothesis we engineered a series of mutants along the length of the IKKγ molecule that could be individually modified with nitroxide spin labels. Subsequent distance measurements using electron paramagnetic resonance spectroscopy combined with molecular modeling and molecular dynamics simulations revealed that IKKγ is a parallel coiled-coil whose response to binding of vFLIP or IKKß is localized twisting/stiffening and not large-scale rearrangements. The coiled-coil comprises N- and C-terminal regions with distinct registers accommodated by a twist: this structural motif is exploited by vFLIP, allowing it to bind and subsequently activate the NF-κB pathway. In vivo assays confirm that NF-κB activation by vFLIP only requires the N-terminal region up to the transition between the registers, which is located directly C-terminal of the vFLIP binding site.

Dimeric chlorite dismutase from the nitrogen-fixing cyanobacterium Cyanothece sp. PCC7425.

It is demonstrated that cyanobacteria (both azotrophic and non-azotrophic) contain heme b oxidoreductases that can convert chlorite to chloride and molecular oxygen (incorrectly denominated chlorite 'dismutase', Cld). Beside the water-splitting manganese complex of photosystem II, this metalloenzyme is the second known enzyme that catalyses the formation of a covalent oxygen-oxygen bond. All cyanobacterial Clds have a truncated N-terminus and are dimeric (i.e. clade 2) proteins. As model protein, Cld from Cyanothece sp. PCC7425 (CCld) was recombinantly produced in Escherichia coli and shown to efficiently degrade chlorite with an activity optimum at pH 5.0 [kcat 1144 ± 23.8 s(-1), KM 162 ± 10.0 µM, catalytic efficiency (7.1 ± 0.6) × 10(6) M(-1) s(-1)]. The resting ferric high-spin axially symmetric heme enzyme has a standard reduction potential of the Fe(III)/Fe(II) couple of -126 ± 1.9 mV at pH 7.0. Cyanide mediates the formation of a low-spin complex with k(on) = (1.6 ± 0.1) × 10(5) M(-1) s(-1) and k(off) = 1.4 ± 2.9 s(-1) (KD ∼ 8.6 µM). Both, thermal and chemical unfolding follows a non-two-state unfolding pathway with the first transition being related to the release of the prosthetic group. The obtained data are discussed with respect to known structure-function relationships of Clds. We ask for the physiological substrate and putative function of these O2 -producing proteins in (nitrogen-fixing) cyanobacteria.

Mechanism of chlorite degradation to chloride and dioxygen by the enzyme chlorite dismutase.

Heme b containing chlorite dismutase (Cld) catalyses the conversion of chlorite to chloride and dioxygen which includes an unusual OO bond formation. This review summarizes our knowledge about the interaction of chlorite with heme enzymes and introduces the biological role, phylogeny and structure of functional chlorite dismutases with differences in overall structure and subunit architecture. The paper sums up the available experimental and computational studies on chlorite degradation by water soluble porphyrin complexes as well as a model based on the active site of Cld. Finally, it reports the available biochemical and biophysical data of Clds from different organisms which allow the presentation of a general reaction mechanism. It includes binding of chlorite to ferric Cld followed by subsequent heterolytic OCl bond cleavage leading to the formation of Compound I and hypochlorite, which finally recombine for production of chloride and O2. The role of the Cld-typical distal arginine in catalysis is discussed together with the pH dependence of the reaction and the role of transiently produced hypochlorite in irreversible inactivation of the enzyme.

Multidomain human peroxidasin 1 is a highly glycosylated and stable homotrimeric high spin ferric peroxidase.

Human peroxidasin 1 (hsPxd01) is a multidomain heme peroxidase that uses bromide as a cofactor for the formation of sulfilimine cross-links. The latter confers critical structural reinforcement to collagen IV scaffolds. Here, hsPxd01 and various truncated variants lacking nonenzymatic domains were recombinantly expressed in HEK cell lines. The N-glycosylation site occupancy and disulfide pattern, the oligomeric structure, and unfolding pathway are reported. The homotrimeric iron protein contains a covalently bound ferric high spin heme per subunit with a standard reduction potential of the Fe(III)/Fe(II) couple of -233 ± 5 mV at pH 7.0. Despite sequence homology at the active site and biophysical properties similar to human peroxidases, the catalytic efficiency of bromide oxidation (kcat/KM(app)) of full-length hsPxd01 is rather low but increased upon truncation. This is discussed with respect to its structure and proposed biosynthetic function in collagen IV cross-linking.

Independent evolution of four heme peroxidase superfamilies.

Four heme peroxidase superfamilies (peroxidase-catalase, peroxidase-cyclooxygenase, peroxidase-chlorite dismutase and peroxidase-peroxygenase superfamily) arose independently during evolution, which differ in overall fold, active site architecture and enzymatic activities. The redox cofactor is heme b or posttranslationally modified heme that is ligated by either histidine or cysteine. Heme peroxidases are found in all kingdoms of life and typically catalyze the one- and two-electron oxidation of a myriad of organic and inorganic substrates. In addition to this peroxidatic activity distinct (sub)families show pronounced catalase, cyclooxygenase, chlorite dismutase or peroxygenase activities. Here we describe the phylogeny of these four superfamilies and present the most important sequence signatures and active site architectures. The classification of families is described as well as important turning points in evolution. We show that at least three heme peroxidase superfamilies have ancient prokaryotic roots with several alternative ways of divergent evolution. In later evolutionary steps, they almost always produced highly evolved and specialized clades of peroxidases in eukaryotic kingdoms with a significant portion of such genes involved in coding various fusion proteins with novel physiological functions.

Structure and heme-binding properties of HemQ (chlorite dismutase-like protein) from Listeria monocytogenes.

Chlorite dismutase-like proteins are structurally closely related to functional chlorite dismutases which are heme b-dependent oxidoreductases capable of reducing chlorite to chloride with simultaneous production of dioxygen. Chlorite dismutase-like proteins are incapable of performing this reaction and their biological role is still under discussion. Recently, members of this large protein family were shown to be involved in heme biosynthesis in Gram-positive bacteria, and thus the protein was renamed HemQ in these organisms. In the present work the structural and heme binding properties of the chlorite dismutase-like protein from the Gram-positive pathogen Listeria monocytogenes (LmCld) were analyzed in order to evaluate its potential role as a regulatory heme sensing protein. The homopentameric crystal structure (2.0Å) shows high similarity to chlorite-degrading chlorite dismutases with an important difference in the structure of the putative substrate and heme entrance channel. In solution LmCld is a stable hexamer able to bind the low-spin ligand cyanide. Heme binding is reversible with KD-values determined to be 7.2µM (circular dichroism spectroscopy) and 16.8µM (isothermal titration calorimetry) at pH 7.0. Both acidic and alkaline conditions promote heme release. Presented biochemical and structural data reveal that the chlorite dismutase-like protein from L. monocytogenes could act as a potential regulatory heme sensing and storage protein within heme biosynthesis.

Interactions of hydrogen sulfide with myeloperoxidase.

BACKGROUND AND PURPOSE: The actions of hydrogen sulfide in human physiology have been extensively studied and, although it is an essential mediator of many biological functions, the underlying molecular mechanisms of its actions are ill-defined. To elucidate the roles of sulfide in inflammation, we have investigated its interactions with human myeloperoxidase (MPO), a major contributor to inflammatory oxidative stress. EXPERIMENTAL APPROACH: The interactions of sulfide and MPO were investigated using electron paramagnetic resonance, electronic circular dichroism, UV-vis and stopped-flow spectroscopies. KEY RESULTS: We found favourable reactions between sulfide and the native-ferric enzyme as well as the MPO redox intermediates, ferrous MPO, compound I and compound II. Sulfide was a potent reversible inhibitor of MPO enzymic activity with an IC50 of 1 µM. In addition, the measured second-order rate constants for the reactions of sulfide with compound I [k = (1.1 ± 0.06) × 10(6) M(-1) s(-1)] and compound II [k = (2.0 ± 0.03) × 10(5) M(-1) s(-1)] suggest that sulfide is a potential substrate for MPO in vivo. CONCLUSION AND IMPLICATIONS: Endogenous levels of sulfide are likely to inhibit the activity of circulating and endothelium-bound MPO. The fully reversible inhibition suggests a mediatory role of sulfide on the oxidant-producing function of the enzyme. Furthermore, the efficient HOCl oxidation of sulfide to give polysulfides (recently recognized as important components of sulfide biology) together with MPO-catalysed sulfide oxidation and the lack of interaction between MPO and sulfide oxidation products, predict a modulatory role of MPO in sulfide signalling.

The structure and regulation of human muscle α-actinin.

The spectrin superfamily of proteins plays key roles in assembling the actin cytoskeleton in various cell types, crosslinks actin filaments, and acts as scaffolds for the assembly of large protein complexes involved in structural integrity and mechanosensation, as well as cell signaling. α-actinins in particular are the major actin crosslinkers in muscle Z-disks, focal adhesions, and actin stress fibers. We report a complete high-resolution structure of the 200 kDa α-actinin-2 dimer from striated muscle and explore its functional implications on the biochemical and cellular level. The structure provides insight into the phosphoinositide-based mechanism controlling its interaction with sarcomeric proteins such as titin, lays a foundation for studying the impact of pathogenic mutations at molecular resolution, and is likely to be broadly relevant for the regulation of spectrin-like proteins.

Quantification of reactive oxygen species generation by photoexcitation of PEGylated quantum dots.

Photocatalytic generation of reactive oxygen species (ROS) from quantum dots (QDs) has been widely reported yet quantitative studies of ROS formation and their quantum yields are lacking. This study investigates the generation of ROS by water soluble PEGylated CdSe/ZnS QDs with red emission. PEGylation of QDs is commonly used to confer water solubility and minimise uptake by organs of the reticuloendothelial system; therefore studies of ROS formation are of biomedical relevance. Using non-photolytic visible wavelength excitation, the superoxide anion radical is shown to be the primary ROS species generated with a quantum efficiency of 0.35%. The yield can be significantly enhanced in the presence of the electron donor, nicotinamide adenine dinucleotide (NADH), as demonstrated by oxygen consumption measurements and electron paramagnetic resonance spectroscopy with in situ illumination. Direct production of singlet oxygen is not detectable from the QDs alone. A comparison is made with ROS generation by the same QDs complexed with a sulfonated phthalocyanine which can generate singlet oxygen via Förster resonance energy transfer between the QDs and the phthalocyanine.

Effects of tribochemical treatments on the integrity of cellulose.

The effects of tribochemical impacts--sonochemical treatments and milling variants--on cellulose were studied, employing multi-detector size exclusion chromatography with group-selective fluorescence labelling ("CCOA method") and diagnostic beta-elimination as the analytical tools. Milling at different temperatures was compared to sonochemical degradation of cellulose by a 24 kHz ultrasound probe system in homogeneous solution. Chain cleavage was generally accompanied by random oxidation of cellulose. The degree of oxidation increased with increasing temperature; at 77K oxidation occurs only to a minor extent. Degradation proceeded towards a final value, the limiting molecular weight (Mw(lim)), beneath which no further decrease of the chain length occurred even at prolonged treatment times. Regardless of the lignin or hemicellulose content of the pulps, the Mw(lim) reached in a specific milling process was largely constant and showed little dependence on the substrate used. The formation of radicals during tribochemical treatments under different conditions and with different substrates was discussed based on theoretical considerations and EPR data. The overall radical content increased with increasing time of milling until a plateau is reached. Here, the mechano-radical content largely depends on the lignin content in the pulp as anticipated. The formation of different radical species and their precursor structures were discussed.

Agaricus meleagris pyranose dehydrogenase: influence of covalent FAD linkage on catalysis and stability.

Pyranose dehydrogenase (PDH) is a monomeric flavoprotein belonging to the glucose-methanol-choline (GMC) family of oxidoreductases. It catalyzes the oxidation of free, non-phosphorylated sugars to the corresponding keto sugars. The enzyme harbors an FAD cofactor that is covalently attached to histidine 103 via an 8α-N(3) histidyl linkage. Our previous work showed that variant H103Y was still able to bind FAD (non-covalently) and perform catalysis but steady-state kinetic parameters for several substrates were negatively affected. In order to investigate the impact of the covalent FAD attachment in Agaricus meleagris PDH in more detail, pre-steady-state kinetics, reduction potential and stability of the variant H103Y in comparison to the wild-type enzyme were probed. Stopped-flow analysis revealed that the mutation slowed down the reductive half-reaction by around three orders of magnitude whereas the oxidative half-reaction was affected only to a minor degree. This was reflected by a decrease in the standard reduction potential of variant H103Y compared to the wild-type protein. The existence of an anionic semiquinone radical in the resting state of both the wild-type and variant H103Y was demonstrated using electron paramagnetic resonance (EPR) spectroscopy and suggested a higher mobility of the cofactor in the variant H103Y. Unfolding studies showed significant negative effects of the disruption of the covalent bond on thermal and conformational stability. The results are discussed with respect to the role of covalently bound FAD in catalysis and stability.

Transiently produced hypochlorite is responsible for the irreversible inhibition of chlorite dismutase.

Chlorite dismutases (Clds) are heme b-containing prokaryotic oxidoreductases that catalyze the reduction of chlorite to chloride with the concomitant release of molecular oxygen. Over time, they are irreversibly inactivated. To elucidate the mechanism of inactivation and investigate the role of the postulated intermediate hypochlorite, the pentameric chlorite dismutase of "Candidatus Nitrospira defluvii" (NdCld) and two variants (having the conserved distal arginine 173 exchanged with alanine and lysine) were recombinantly produced in Escherichia coli. Exchange of the distal arginine boosts the extent of irreversible inactivation. In the presence of the hypochlorite traps methionine, monochlorodimedone, and 2-[6-(4-aminophenoxy)-3-oxo-3H-xanthen-9-yl]benzoic acid, the extent of chlorite degradation and release of molecular oxygen is significantly increased, whereas heme bleaching and oxidative modifications of the protein are suppressed. Among other modifications, hypochlorite-mediated formation of chlorinated tyrosines is demonstrated by mass spectrometry. The data obtained were analyzed with respect to the proposed reaction mechanism for chlorite degradation and its dependence on pH. We discuss the role of distal Arg173 by keeping hypochlorite in the reaction sphere for O-O bond formation.

Mechanism of reaction of chlorite with mammalian heme peroxidases.

This study demonstrates that heme peroxidases from different superfamilies react differently with chlorite. In contrast to plant peroxidases, like horseradish peroxidase (HRP), the mammalian counterparts myeloperoxidase (MPO) and lactoperoxidase (LPO) are rapidly and irreversibly inactivated by chlorite in the micromolar concentration range. Chlorite acts as efficient one-electron donor for Compound I and Compound II of MPO and LPO and reacts with the corresponding ferric resting states in a biphasic manner. The first (rapid) phase is shown to correspond to the formation of a MPO-chlorite high-spin complex, whereas during the second (slower) phase degradation of the prosthetic group was observed. Cyanide, chloride and hydrogen peroxide can block or delay heme bleaching. In contrast to HRP, the MPO/chlorite system does not mediate chlorination of target molecules. Irreversible inactivation is shown to include heme degradation, iron release and decrease in thermal stability. Differences between mammalian peroxidases and HRP are discussed with respect to differences in active site architecture and heme modification.

Manipulating conserved heme cavity residues of chlorite dismutase: effect on structure, redox chemistry, and reactivity.

Chlorite dismutases (Clds) are heme b containing oxidoreductases that convert chlorite to chloride and molecular oxygen. In order to elucidate the role of conserved heme cavity residues in the catalysis of this reaction comprehensive mutational and biochemical analyses of Cld from "Candidatus Nitrospira defluvii" (NdCld) were performed. Particularly, point mutations of the cavity-forming residues R173, K141, W145, W146, and E210 were performed. The effect of manipulation in 12 single and double mutants was probed by UV-vis spectroscopy, spectroelectrochemistry, pre-steady-state and steady-state kinetics, and X-ray crystallography. Resulting biochemical data are discussed with respect to the known crystal structure of wild-type NdCld and the variants R173A and R173K as well as the structures of R173E, W145V, W145F, and the R173Q/W146Y solved in this work. The findings allow a critical analysis of the role of these heme cavity residues in the reaction mechanism of chlorite degradation that is proposed to involve hypohalous acid as transient intermediate and formation of an O═O bond. The distal R173 is shown to be important (but not fully essential) for the reaction with chlorite, and, upon addition of cyanide, it acts as a proton acceptor in the formation of the resulting low-spin complex. The proximal H-bonding network including K141-E210-H160 keeps the enzyme in its ferric (E°' = -113 mV) and mainly five-coordinated high-spin state and is very susceptible to perturbation.

A stable bacterial peroxidase with novel halogenating activity and an autocatalytically linked heme prosthetic group.

Reconstructing the phylogenetic relationships of the main evolutionary lines of the mammalian peroxidases lactoperoxidase and myeloperoxidase revealed the presence of novel bacterial heme peroxidase subfamilies. Here, for the first time, an ancestral bacterial heme peroxidase is shown to possess a very high bromide oxidation activity (besides conventional peroxidase activity). The recombinant protein allowed monitoring of the autocatalytic peroxide-driven formation of covalent heme to protein bonds. Thereby, the high spin ferric rhombic heme spectrum became similar to lactoperoxidase, the standard reduction potential of the Fe(III)/Fe(II) couple shifted to more positive values (-145 ± 10 mV at pH 7), and the conformational and thermal stability of the protein increased significantly. We discuss structure-function relationships of this new peroxidase in relation to its mammalian counterparts and ask for its putative physiological role.

Tocopheramines and tocotrienamines as antioxidants: ESR spectroscopy, rapid kinetics and DFT calculations.

Tocopheramines (TNH2) and tocotrienamines (T3NH2) are analogues of tocopherols (TOH) and tocotrienols in which phenolic OH is replaced by NH2. It was shown in previous studies that TNH2 and T3NH2 act as potent antioxidants. In this study we compared the one-electron oxidation of TNH2/T3NH2 by diphenyl picryl hydrazyl (DPPH) and galvinoxyl (GOX) radicals with the one of α-TOH as a reference compound using ESR spectroscopy, stopped flow spectrophotometry and density functional theory (DFT) calculations. ESR spectroscopy revealed the presence of tocopheramine radicals during electrochemical oxidation of α-TNH2. Kinetic measurements demonstrated that in apolar n-hexane TNH2/T3NH2 derivatives reacted two to three orders of magnitude slower than α-TOH with the model radicals. DFT calculations indicated that this correlates well with the higher bond dissociation energy (BDE) for N-H in TNH2 than for O-H in α-TOH in pure H-atom transfer (HAT). In the more polar medium ethanol TNH2/T3NH2 derivatives partially reacted faster than α-TOH depending on the reaction partner. DFT calculations suggest that this is due to reaction mechanisms alternative to HAT. According to thermochemistry data sequential proton loss and electron transfer (SPLET) is more favored for α-TOH in ethanol than for TNH2. Therefore, for TNH2 a contribution of the alternative mechanism of sequential electron transfer-proton transfer (SET-PT) could be a possible explanation. These data show that the antioxidant reactivity strongly depends on the structure, reaction partners and environment. According to these findings TNH2/T3NH2 should be superior as antioxidants over α-TOH in polar head group regions of membranes but not in the apolar core of lipid bilayers.