Was hydrogen peroxide present before the arrival of oxygenic photosynthesis? The important role of iron(II) in the Archean ocean.

We address the chemical/biological history of H2O2 back at the times of the Archean eon (2.5-3.9 billion years ago (Gya)). During the Archean eon the pO2 was million-fold lower than the present pO2, starting to increase gradually from 2.3 until 0.6 Gya, when it reached ca. 0.2 bar. The observation that some anaerobic organisms can defend themselves against O2 has led to the view that early organisms could do the same before oxygenic photosynthesis had developed at about 3 Gya. This would require the anaerobic generation of H2O2, and here we examine the various mechanisms which were suggested in the literature for this. Given the concentration of Fe2+ at 20-200 µM in the Archean ocean, the estimated half-life of H2O2 is ca. 0.7 s. The oceanic H2O2 concentration was practically zero. We conclude that early organisms were not exposed to H2O2 before the arrival of oxygenic photosynthesis.

Rust never sleeps: The continuing story of the Iron Bolt.

Since 1981, Gordon Research Conferences have been held on the topic of Oxygen Radicals on a biennial basis, to highlight and discuss the latest cutting edge research in this area. Since the first meeting, one special feature of this conference has been the awarding of the so-called Iron Bolt, an award that started in jest but has gained increasing reputation over the years. Since no written documentation exists for this Iron Bolt award, this perspective serves to overview the history of this unusual award, and highlights various experiences of previous winners of this "prestigious" award and other interesting anecdotes.

Low-Temperature Trapping of Intermediates in the Reaction of NO• with O2.

The autoxidation of NO• was studied in glass-like matrices of 2-methylbutane at 110 K and in a 8:3 v/v mixture of 2,2-dimethylbutane and n-pentane (rigisolve) at 80-90 K, by letting gaseous NO• diffuse into these solvents that were saturated with O2. In 2-methyllbutane, we observed a red compound. However, in rigisolve at 85-90 K, a bright yellow color appears that turns red when the sample is warmed by 10-20 K. The new yellow compound is a precursor of the red one and also diamagnetic. The UV-vis spectrum of the yellow compound contains a band which resembles that present in ONOO-. Because the red and yellow intermediates are not paramagnetic, we postulate that O═N-O-O• is in close contact with NO•, or with another O═N-O-O•. Diffusion of gaseous O2 into rigisolve saturated with NO• does not produce a color; however, a weak EPR signal (g = 2.010) is observed. This signal most likely indicates the presence of ONOO•. These findings complement our earlier observation of a red color at low temperatures and the presence of ONOO• in the gas phase (Galliker, B.; Kissner, R.; Nauser, T.; Koppenol, W. H. Chem. Eur. J. 2009, 15, 6161-6168), and they indicate that the termolecular autoxidation of nitrogen monoxide proceeds via the intermediate ONOO• and not via N2O2.

Redox properties and activity of iron-citrate complexes: evidence for redox cycling.

Iron in iron overload disease is present as non-transferrin-bound iron, consisting of iron, citrate, and albumin. We investigated the redox properties of iron citrate by electrochemistry, by the kinetics of its reaction with ascorbate, by ESR, and by analyzing the products of reactions of ascorbate with iron citrate complexes in the presence of H2O2 with 4-hydroxybenzoic acid as a reporter molecule for hydroxylation. We report -0.03 V < E°' > +0.01 V for the (Fe(3+)-cit/Fe(2+)-cit) couple. The first step in the reaction of iron citrate with ascorbate is the rapid formation of mixed complexes of iron with citrate and ascorbate, followed by slow reduction to Fe(2+)-citrate with k = ca. 3 M(-1) s(-1). The ascorbyl radical is formed by iron citrate oxidation of Hasc(-) with k = ca. 0.02 M(-1) s(-1); the majority of the ascorbyl radical formed is sequestered by complexation with iron and remains EPR silent. The hydroxylation of 4-hydroxybenzoic acid driven by the Fenton reduction of iron citrate by ascorbate in the presence of H2O2 proceeds in three phases: the first phase, which is independent of the presence of O2, is revealed as a nonzero intercept that reflects the rapid reaction of accumulated Fe(2+) with H2O2; the intermediate oxygen-dependent phase fits a first-order accumulation of product with k = 5 M(-1) s(-1) under aerobic and k = 13 M(-1) s(-1) under anaerobic conditions; the slope of the final linear phase is ca. k = 5 × 10(-2) M(-1) s(-1) under both aerobic and anaerobic conditions. Product yields under aerobic conditions are greater than predicted from the initial concentration of iron, but they are less than predicted for continuous redox cycling in the presence of excess ascorbate. The ongoing formation of hydroxylated product supports slow redox cycling by iron citrate. Thus, when H2O2 is available, iron-citrate complexes may contribute to pathophysiological manifestations of iron overload diseases.

Iron(II) binding by cereal beta-glucan.

Beta-glucan is a dietary fiber, which possesses several health benefits, such as cholesterol lowering, however this fiber is easily degraded in the presence of Fenton reagents. In the present study, the iron binding capacity of oat beta-glucan and barley beta-glucan was evaluated by investigating the kinetics of the Fenton reaction at pH 2.7 and 4.7 using stopped flow spectroscopy. The rate constant of the Fenton reaction is not affected by the presence of the beta-glucans in a solution pH 2.7, hence none of the beta-glucans bind iron(II) at this pH. However, at pH 4.7, the kinetics of the Fenton reaction vary between acetate buffer (k=2.8×10(2)M(-1)s(-1)), barley beta-glucan (k=2.2×10(2)M(-1)s(-1)) and oat beta-glucan (k=1.2×10(2)M(-1)s(-1)), which demonstrates that barley beta-glucan and oat beta-glucan form complexes with iron(II). Moreover, oat beta-glucan shows a stronger affinity for iron(II) than barley beta-glucan, and may thereby reduce the formation of hydroxyl radicals and diminish the rate of viscosity loss of the oat beta-glucan solution, as shown by the ESR and rheological data. The results presented in this study suggest that cereal beta-glucans can potentially reduce the bioavailability of iron.

Protein thiyl radical reactions and product formation: a kinetic simulation.

Protein thiyl radicals are important intermediates generated in redox processes of thiols and disulfides. Thiyl radicals efficiently react with glutathione and ascorbate, and the common notion is that these reactions serve to eliminate thiyl radicals before they can enter potentially hazardous processes. However, over the past years increasing evidence has been provided for rather efficient intramolecular hydrogen transfer processes of thiyl radicals in proteins and peptides. Based on rate constants published for these processes, we have performed kinetic simulations of protein thiyl radical reactivity. Our simulations suggest that protein thiyl radicals enter intramolecular hydrogen transfer reactions to a significant extent even under physiologic conditions, i.e., in the presence of 30 µM oxygen, 1 mM ascorbate, and 10 mM glutathione. At lower concentrations of ascorbate and glutathione, frequently observed when tissue is exposed to oxidative stress, the extent of irreversible protein thiyl radical-dependent protein modification increases.

Why selenocysteine replaces cysteine in thioredoxin reductase: a radical hypothesis.

Thioredoxin reductases, important biological redox mediators for two-electron transfers, contain either 2 cysteines or a cysteine (Cys) and a selenocysteine (Sec) at the active site. The incorporation of Sec is metabolically costly, and therefore surprising. We provide here a rationale: in the case of an accidental one-electron transfer to a S-S or a S-Se bond during catalysis, a thiyl or a selanyl radical, respectively would be formed. The thiyl radical can abstract a hydrogen from the protein backbone, which subsequently leads to the inactivation of the protein. In contrast, a selanyl radical will not abstract a hydrogen. Therefore, formation of Sec radicals in a GlyCysSecGly active site will less likely result in the destruction of a protein compared to a GlyCysCysGly active site.

Rapid reaction of superoxide with insulin-tyrosyl radicals to generate a hydroperoxide with subsequent glutathione addition.

Tyrosine (Tyr) residues are major sites of radical generation during protein oxidation. We used insulin as a model to study the kinetics, mechanisms, and products of the reactions of radiation-induced or enzyme-generated protein-tyrosyl radicals with superoxide to demonstrate the feasibility of these reactions under oxidative stress conditions. We found that insulin-tyrosyl radicals combined to form dimers, mostly via the tyrosine at position 14 on the α chain (Tyr14). However, in the presence of superoxide, dimerization was largely outcompeted by the reaction of superoxide with insulin-tyrosyl radicals. Using pulse radiolysis, we measured a second-order rate constant for the latter reaction of (6±1) × 10(8) M(-1) s(-1) at pH 7.3, representing the first measured rate constant for a protein-tyrosyl radical with superoxide. Mass-spectrometry-based product analyses revealed the addition of superoxide to the insulin-Tyr14 radical to form the hydroperoxide. Glutathione efficiently reduced the hydroperoxide to the corresponding monoxide and also subsequently underwent Michael addition to the monoxide to give a diglutathionylated protein adduct. Although much slower, conjugation of the backbone amide group can form a bicyclic Tyr-monoxide derivative, allowing the addition of only one glutathione molecule. These findings suggest that Tyr-hydroperoxides should readily form on proteins under oxidative stress conditions where protein radicals and superoxide are both generated and that these should form addition products with thiol compounds such as glutathione.

The kinetics of the reaction of nitrogen dioxide with iron(II)- and iron(III) cytochrome c.

The reactions of NO2 with both oxidized and reduced cytochrome c at pH 7.2 and 7.4, respectively, and with N-acetyltyrosine amide and N-acetyltryptophan amide at pH 7.3 were studied by pulse radiolysis at 23 °C. NO2 oxidizes N-acetyltyrosine amide and N-acetyltryptophan amide with rate constants of (3.1±0.3)×10(5) and (1.1±0.1)×10(6) M(-1) s(-1), respectively. With iron(III)cytochrome c, the reaction involves only its amino acids, because no changes in the visible spectrum of cytochrome c are observed. The second-order rate constant is (5.8±0.7)×10(6) M(-1) s(-1) at pH 7.2. NO2 oxidizes iron(II)cytochrome c with a second-order rate constant of (6.6±0.5)×10(7) M(-1) s(-1) at pH 7.4; formation of iron(III)cytochrome c is quantitative. Based on these rate constants, we propose that the reaction with iron(II)cytochrome c proceeds via a mechanism in which 90% of NO2 oxidizes the iron center directly-most probably via reaction at the solvent-accessible heme edge-whereas 10% oxidizes the amino acid residues to the corresponding radicals, which, in turn, oxidize iron(II). Iron(II)cytochrome c is also oxidized by peroxynitrite in the presence of CO2 to iron(III)cytochrome c, with a yield of ~60% relative to peroxynitrite. Our results indicate that, in vivo, NO2 will attack preferentially the reduced form of cytochrome c; protein damage is expected to be marginal, the consequence of formation of amino acid radicals on iron(III)cytochrome c.

The complex interplay of iron metabolism, reactive oxygen species, and reactive nitrogen species: insights into the potential of various iron therapies to induce oxidative and nitrosative stress.

Production of minute concentrations of superoxide (O2(*-)) and nitrogen monoxide (nitric oxide, NO*) plays important roles in several aspects of cellular signaling and metabolic regulation. However, in an inflammatory environment, the concentrations of these radicals can drastically increase and the antioxidant defenses may become overwhelmed. Thus, biological damage may occur owing to redox imbalance-a condition called oxidative and/or nitrosative stress. A complex interplay exists between iron metabolism, O2(*-), hydrogen peroxide (H2O2), and NO*. Iron is involved in both the formation and the scavenging of these species. Iron deficiency (anemia) (ID(A)) is associated with oxidative stress, but its role in the induction of nitrosative stress is largely unclear. Moreover, oral as well as intravenous (iv) iron preparations used for the treatment of ID(A) may also induce oxidative and/or nitrosative stress. Oral administration of ferrous salts may lead to high transferrin saturation levels and, thus, formation of non-transferrin-bound iron, a potentially toxic form of iron with a propensity to induce oxidative stress. One of the factors that determine the likelihood of oxidative and nitrosative stress induced upon administration of an iv iron complex is the amount of labile (or weakly-bound) iron present in the complex. Stable dextran-based iron complexes used for iv therapy, although they contain only negligible amounts of labile iron, can induce oxidative and/or nitrosative stress through so far unknown mechanisms. In this review, after summarizing the main features of iron metabolism and its complex interplay with O2(*-), H2O2, NO*, and other more reactive compounds derived from these species, the potential of various iron therapies to induce oxidative and nitrosative stress is discussed and possible underlying mechanisms are proposed. Understanding the mechanisms, by which various iron formulations may induce oxidative and nitrosative stress, will help us develop better tolerated and more efficient therapies for various dysfunctions of iron metabolism.

Hydrogen exchange equilibria in thiols.

Cysteine, cysteinyl-glycine, glutathione, phenylalanyl-cysteinyl-glycine, and histidyl-cysteinyl-glycine were dissolved in acidic and neutral D(2)O in the presence of the radical generator 2,2'-azobis(2-methylpropionamidine) dihydrochloride and radical mediator compounds (benzyl alcohol and 2-propanol). An exchange of H-atoms by D-atoms took place in these peptides due to intramolecular H-abstraction equilibria. NMR measurements allow one to follow the extent of H-D exchanges and to identify the sites where these exchanges take place. Significant exchanges occur in acidic media in GSH at positions Glu-ß and Glu-γ, in Phe-Cys-Gly at positions Phe ortho, Phe-ß, Cys-α, Cys-ß, and Gly-α, and in His-Cys-Gly at positions His H1, His H2, His ß, Cys ß, and Gly α. In neutral media, exchanges occur in Cys-Gly at position Cys ß and in GSH at position Cys α. Phe-Cys-Gly and His-Cys-Gly were not examined in neutral media. Sites participating in the radical exchange equilibria are highly dependent on structure and pH; the availability of electron density in the form of lone pairs appears to increase the extent of exchange. Interestingly, and unexpectedly, 2D NMR experiments show that GSH rearranges itself in acidic solution: the signals shift, but their patterns do not change. The formation of a thiolactone from Gly and Cys residues matches the changes observed.

Reversible hydrogen transfer reactions in thiyl radicals from cysteine and related molecules: absolute kinetics and equilibrium constants determined by pulse radiolysis.

The mercapto group of cysteine (Cys) is a predominant target for oxidative modification, where one-electron oxidation leads to the formation of Cys thiyl radicals, CysS(•). These Cys thiyl radicals enter 1,2- and 1,3-hydrogen transfer reactions, for which rate constants are reported in this paper. The products of these 1,2- and 1,3-hydrogen transfer reactions are carbon-centered radicals at position C(3) (α-mercaptoalkyl radicals) and C(2) ((•)C(α) radicals) of Cys, respectively. Both processes can be monitored separately in Cys analogues such as cysteamine (CyaSH) and penicillamine (PenSH). At acidic pH, thiyl radicals from CyaSH permit only the 1,2-hydrogen transfer according to equilibrium 12, (+)H(3)NCH(2)CH(2)S(• )⇌ (+)H(3)NCH(2)(•)CH-SH, where rate constants for forward and reverse reaction are k(12) ≈ 10(5) s(-1) and k(-12) ≈ 1.5 × 10(5)s(-1), respectively. In contrast, only the 1,3-hydrogen transfer is possible for thiyl radicals from PenSH according to equilibrium 14, ((+)H(3)N/CO(2)H)C(α)-C(CH(3))(2)-S(•) ⇌ ((+)H(3)N/CO(2)H)(•)C(α)-C(CH(3))(2)-SH, where rate constants for the forward and the reverse reaction are k(14) = 8 × 10(4) s(-1) and k(-14) = 1.4 × 10(6) s(-1). The (•)C(α) radicals from PenSH and Cys have the additional opportunity for ß-elimination of HS(•)/S(•-), which proceeds with k(39) ≈ (3 ± 1) × 10(4) s(-1) from (•)C(α) radicals from PenSH and k(-34) ≈ 5 × 10(3) s(-1) from (•)C(α) radicals from Cys. The rate constants quantified for the 1,2- and 1,3-hydrogen transfer reactions can be used as a basis to calculate similar processes for Cys thiyl radicals in proteins, where hydrogen transfer reactions, followed by the addition of oxygen, may lead to the irreversible modification of target proteins.

Why do proteins use selenocysteine instead of cysteine?

Selenocysteine is present in a variety of proteins and catalyzes the oxidation of thiols to disulfides and the reduction of disulfides to thiols. Here, we compare the kinetic and thermodynamic properties of cysteine with its selenium-containing analogon, selenocysteine. Reactions of simple selenols at pH 7 are up to four orders of magnitude faster than their sulfur analogs, depending on reaction type. In redox-related proteins, the use of selenium instead of sulfur can be used to tune electrode, or redox, potentials. Selenocysteine could also have a protective effect in proteins because its one-electron oxidized product, the selanyl radical, is not oxidizing enough to modify or destroy proteins, whereas the cysteine-thiyl radical can do this very rapidly.

Otto Warburg's contributions to current concepts of cancer metabolism.

Otto Warburg pioneered quantitative investigations of cancer cell metabolism, as well as photosynthesis and respiration. Warburg and co-workers showed in the 1920s that, under aerobic conditions, tumour tissues metabolize approximately tenfold more glucose to lactate in a given time than normal tissues, a phenomenon known as the Warburg effect. However, this increase in aerobic glycolysis in cancer cells is often erroneously thought to occur instead of mitochondrial respiration and has been misinterpreted as evidence for damage to respiration instead of damage to the regulation of glycolysis. In fact, many cancers exhibit the Warburg effect while retaining mitochondrial respiration. We re-examine Warburg's observations in relation to the current concepts of cancer metabolism as being intimately linked to alterations of mitochondrial DNA, oncogenes and tumour suppressors, and thus readily exploitable for cancer therapy.

Distance-dependent diffusion-controlled reaction of •NO and O2•- at chemical equilibrium with ONOO-.

The fast reaction of (•)NO and O(2)(•-) to give ONOO(-) has been extensively studied at irreversible conditions, but the reasons for the wide variations in observed forward rate constants (3.8 ≤ k(f) ≤ 20 × 10(9) M(-1) s(-1)) remain unexplained. We characterized the diffusion-dependent aqueous (pH > 12) chemical equilibrium of the form (•)NO + O(2)(•-) = ONOO(-) with respect to its dependence on temperature, viscosity, and [ONOO(-)](eq) by determining [ONOO(-)](eq) and [(•)NO](eq). The equilibrium forward reaction rate constant (k(f)(eq)) has negative activation energy, in contrast to that found under irreversible conditions. In contradiction to the law of mass action, we demonstrate that the equilibrium constant depends on ONOO(-) concentration. Therefore, a wide range of k(f)(eq) values could be derived (7.5-21 × 10(9) M(-1) s(-1)). Of general interest, the variations in k(f) can thus be explained by its dependence on the distance between ONOO(-) particles (sites of generation of (•)NO and O(2)(•-)).

Hydrogen exchange equilibria in glutathione radicals: rate constants.

The reduction of oxidized glutathione GSSG by hydrated electrons and hydrogen atoms to form GSSG•⁻ is quantitative. The radical anion dissociates into GS• and GS⁻, and the S-centered radical subsequently abstracts a hydrogen intramolecularly. We observe sequential development of UV absorbance signatures that indicate the formation of both α- and ß-carbon-centered radicals. From experiments performed at pH 2 and pH 11.8, we determined forward and reverse rate constants for the overall equilibrium between sulfur-centered and carbon-centered radicals: k(forward) = 3·105 s⁻¹, k(reverse) = 7·105 s⁻¹, and K = 0.4. Furthermore, on the basis of the differences between the kinetics traces at 240 and 280 nm, we estimate that α- and ß-carbon-centered radicals are formed at a surprising ratio of 1:3. The ratios found at pH 2 also apply to pH 7, with the conclusion that the equilibrium ratio of S-centered:ß-centered:α-centered radicals is, very approximately, 8:3:1. The formation of carbon-centered radicals could lead to irreversible damage in proteins via the formation of carbon-carbon bonds or backbone fragmentation.

Selenium and sulfur in exchange reactions: a comparative study.

Cysteamine reduces selenocystamine to form hemiselenocystamine and then cystamine. The rate constants are k(1) = 1.3 × 10(5) M(-1) s(-1); k(-1) = 2.6 × 10(7) M(-1) s(-1); k(2) = 11 M(-1) s(-1); and k(-2) = 1.4 × 10(3) M(-1) s(-1), respectively. Rate constants for reactions of cysteine/selenocystine are similar. Reaction rates of selenium as a nucleophile and as an electrophile are 2-3 and 4 orders of magnitude higher, respectively, than those of sulfur. Sulfides and selenides are comparable as leaving groups.

Reduction of protein radicals by GSH and ascorbate: potential biological significance.

The oxidation of proteins and other macromolecules by radical species under conditions of oxidative stress can be modulated by antioxidant compounds. Decreased levels of the antioxidants glutathione and ascorbate have been documented in oxidative stress-related diseases. A radical generated on the surface of a protein can: (1) be immediately and fully repaired by direct reaction with an antioxidant; (2) react with dioxygen to form the corresponding peroxyl radical; or (3) undergo intramolecular long range electron transfer to relocate the free electron to another amino acid residue. In pulse radiolysis studies, in vitro production of the initial radical on a protein is conveniently made at a tryptophan residue, and electron transfer often leads ultimately to residence of the unpaired electron on a tyrosine residue. We review here the kinetics data for reactions of the antioxidants glutathione, selenocysteine, and ascorbate with tryptophanyl and tyrosyl radicals as free amino acids in model compounds and proteins. Glutathione repairs a tryptophanyl radical in lysozyme with a rate constant of (1.05±0.05)×10(5) M(-1) s(-1), while ascorbate repairs tryptophanyl and tyrosyl radicals ca. 3 orders of magnitude faster. The in vitro reaction of glutathione with these radicals is too slow to prevent formation of peroxyl radicals, which become reduced by glutathione to hydroperoxides; the resulting glutathione thiyl radical is capable of further radical generation by hydrogen abstraction. Although physiologically not significant, selenoglutathione reduces tyrosyl radicals as fast as ascorbate. The reaction of protein radicals formed on insulin, ß-lactoglobulin, pepsin, chymotrypsin and bovine serum albumin with ascorbate is relatively rapid, competes with the reaction with dioxygen, and the relatively innocuous ascorbyl radical is formed. On the basis of these kinetics data, we suggest that reductive repair of protein radicals may contribute to the well-documented depletion of ascorbate in living organisms subjected to oxidative stress.