Kinetic studies of iron deposition catalyzed by recombinant human liver heavy and light ferritins and Azotobacter vinelandii bacterioferritin using O2 and H2O2 as oxidants

The discrepancy between predicted and measured H(2)O(2) formation during iron deposition with recombinant heavy human liver ferritin (rHF) was attributed to reaction with the iron protein complex [Biochemistry 40 (2001) 10832-10838]. This proposal was examined by stopped-flow kinetic studies and analysis for H(2)O(2) production using (1) rHF, and Azotobacter vinelandii bacterial ferritin (AvBF), each containing 24 identical subunits with ferroxidase centers; (2) site-altered rHF mutants with functional and dysfunctional ferroxidase centers; and (3) recombinant human liver light ferritin (rLF), containing no ferroxidase center. For rHF, nearly identical pseudo-first-order rate constants of 0.18 s(-1) at pH 7.5 were measured for Fe(2+) oxidation by both O(2) and H(2)O(2), but for rLF, the rate with O(2) was 200-fold slower than that for H(2)O(2) (k = 0.22 s(-1)). A Fe(2+)/O(2) stoichiometry near 2.4 was measured for rHF and its site altered forms, suggesting formation of H(2)O(2). Direct measurements revealed no H(2)O(2) free in solution 0.5-10 min after all Fe(2+) was oxidized at pH 6.5 or 7.5. These results are consistent with initial H(2)O(2) formation, which rapidly reacts in a secondary reaction with unidentified solution components. Using measured rate constants for rHF, simulations showed that steady-state H(2)O(2) concentrations peaked at 14 muM at approximately 600 ms and decreased to zero at 10-30 s. rLF did not produce measurable H(2)O(2) but apparently conducted the secondary reaction with H(2)O(2). Fe(2+)/O(2) values of 4.0 were measured for AvBF. Stopped-flow measurements with AvBF showed that both H(2)O(2) and O(2) react at the same rate (k = 0.34 s(-1)), that is faster than the reactions with rHF. Simulations suggest that AvBF reduces O(2) directly to H(2)O without intermediate H(2)O(2) formation.     

Hydrogen peroxide formation during iron deposition in horse spleen ferritin using O2 as an oxidant

The reaction of Fe2+ with O2 in the presence of horse spleen ferritin (HoSF) results in deposition of FeOH3 into the hollow interior of HoSF. This reaction was examined at low Fe2+/HoSF ratios (5-100) under saturating air at pH 6.5-8.0 to determine if H2O2 is a product of the iron deposition reaction. Three methods specific for H2O2 detection were used to assess H2O2 formation: (1) a fluorometric method with emission at 590 nm, (2) an optical absorbance method based on the reaction H2O2 + 3I- + 2H+ = I3- + 2H2O monitored at 340 nm for I3- formation, and (3) a differential pulsed electrochemical method that measures O2 and H2O2 concentrations simultaneously. Detection limits of 0.25, 2.5, and 5.0 microM H2O2 were determined for the three methods, respectively. Under constant air-saturation conditions (20% O2) and for a 5-100 Fe2+/HoSF ratio, Fe2+ was oxidized and the resulting Fe3+ was deposited within HoSF but no H2O2 was detected as predicted by the reaction 2Fe2+ + O2 + 6H2O = 2Fe(OH)3 + H2O2 + 4H+. Two other sets of conditions were also examined: one with excess but nonsaturating O2 and another with limiting O2. No H2O2 was detected in either case. The absence of H2O2 formation under these same conditions was confirmed by microcoulometric measurements. Taken together, the results show that under low iron loading conditions (5-100 Fe2+/HoSF ratio), H2O2 is not produced during iron deposition into HoSF using O2 as an oxidant. This conclusion is inconsistent with previous, carefully conducted stoichiometric and kinetic measurements [Xu, B., and Chasteen, N. D. (1991) J. Biol. Chem. 266, 19965], predicting that H2O2 is a quantitative product of the iron deposition reaction with O2 as an oxidant, even though it was not directly detected. Possible explanations for these conflicting results are considered.     

Kinetic studies of iron deposition in horse spleen ferritin using O2 as oxidant

An optical flow cell provided a means to conveniently measure the rate of successive Fe(2+) oxidation reactions catalyzed by horse spleen ferritin (HoSF) to determine if both ferroxidase and mineral core Fe(2+) oxidation reactions occur. The oxygen concentration and pH were held constant and multiple additions of Fe(2+)/HoSF ratios of 1, 10, 100, 150, 250 and 400 were conducted, creating core sizes ranging from 12 to 2800. During these oxidations, the absence of nonspecific Fe(OH)(3) formation and the presence (>95%) of Fe(OH)(3) deposited within the core of HoSF demonstrated the validity of monitoring iron deposition into HoSF by this procedure. Initial rates for oxidation of 5-50 Fe(2+)/HoSF established that the reaction is overall first order in Fe(2+) concentration. However, when full progress curves were analyzed at a variety of Fe(2+)/HoSF ratios, two first-order reactions (k(1) approximately 0.035 s(-1) and k(2) approximately 0.007 s(-1)) were found to contribute to the overall Fe(2+) oxidation reaction. The proportion of the fast reaction increased with increasing Fe(2+)/HoSF ratio until at approximately 400, it was the dominant reaction. For the Fe(2+)/HoSF ratios examined, the overall rate of iron deposition is independent of the size of the mineral core, a result suggesting that an increasing mineral core size does not enhance the rate of Fe(2+) oxidation. Comparison of successive additions of 1.0 Fe(2+)/HoSF showed that oxidation of the first 8-10 Fe(2+) produced a Fe(III) species with a lower molar absorptivity per Fe(III) than that of the bulk core. Measurement of the H(+)/Fe(2+) ratio confirmed this difference in behavior by giving an H(+)/Fe(2+) ratio of approximately 1.0 below and 2.0 for ratios >30 Fe(2+)/HoSF. The faster reaction was attributed to ferroxidase catalysis and the slow reaction to nonspecific ferroxidase activity of the HoSF protein shell.     

Iron oxidation chemistry in ferritin. Increasing Fe/O2 stoichiometry during core formation

The origin of previously observed variations in stoichiometry of iron oxidation during the oxidative deposition of iron in ferritin has been poorly understood. Knowledge of the stoichiometry of Fe(II) oxidation by O2 is essential to establishing the mechanism of iron core formation. In the present work, the amount of Fe(II) oxidized was measured by M?ssbauer spectrometry and the O2 consumed by mass spectrometry. The number of protons produced in the reaction was measured by "pH stat" titration and hydrogen peroxide production by the effect of the enzyme catalase on the measured stoichiometry. For protein samples containing low levels of iron (24 Fe(II)/protein) the stoichiometry was found to be 1.95 +/- 0.18 Fe(II)/O2 with H2O2 being a product, viz. Equation 1. 2Fe2+ + O2 + 4H2O----2FeOOH + H2O2 + 4H+ (1) EPR spin trapping experiments showed no evidence of superoxide radical formation. The stoichiometry markedly increased with additional iron (240-960 Fe/protein), to a value of 4 Fe(II)/O2 as in Equation 2. 4Fe2+ + O2 + 6H2O----4FeOOH + 8H+ (2) As the iron core is progressively laid down, the mechanism of iron oxidation changes from a protein dominated process with H2O2 being the primary product of O2 reduction to a mineral surface dominated process where H2O is the primary product. These results emphasize the importance of the apoferritin shell in facilitating iron oxidation in the early stage of iron deposition prior to significant development of the polynuclear iron core.     

Paired Bacillus anthracis Dps (mini-ferritin) have different reactivities with peroxide

Dps (DNA protection during starvation) proteins, mini-ferritins in the ferritin superfamily, catalyze Fe(2+)/H(2)O(2)/O(2) reactions and make minerals inside protein nanocages to minimize radical oxygen-chemistry (metal/osmotic/temperature/nutrient/oxidant) and sometimes to confer virulence. Paired Dps proteins in Bacillus, rare in other bacteria, have 60% sequence identity. To explore functional differences in paired Bacilli Dps protein, we measured ferroxidase activity and DNA protection (hydroxyl radical) for Dps protein dodecamers from Bacillus anthracis (Ba) since crystal structures and iron mineralization (iron-stain) were known. The self-assembled (200 kDa) Ba Dps1 (Dlp-1) and Ba Dps2 (Dlp-2) proteins had similar Fe(2+)/O(2) kinetics, with space for minerals of 500 iron atoms/protein, and protected DNA. The reactions with Fe(2+) were novel in several ways: 1) Ba Dps2 reactions (Fe(2+)/H(2)O(2)) proceeded via an A(650 nm) intermediate, with similar rates to maxi-ferritins (Fe(2+)/O(2)), indicating a new Dps protein reaction pathway, 2) Ba Dps2 reactions (Fe(2+)/O(2) versus Fe(2+)/O(2) + H(2)O(2)) differed 3-fold contrasting with Escherichia coli Dps reactions, with 100-fold differences, and 3) Ba Dps1, inert in Fe(2+)/H(2)O(2) catalysis, inhibited protein-independent Fe(2+)/H(2)O(2) reactions. Sequence similarities between Ba Dps1 and Bacillus subtilis DpsA (Dps1), which is regulated by general stress factor (SigmaB) and Fur, and between Ba Dps2 and B. subtilis MrgA, which is regulated by H(2)O(2) (PerR), suggest the function of Ba Dps1 is iron sequestration and the function of Ba Dps2 is H(2)O(2) destruction, important in host/pathogen interactions. Destruction of H(2)O(2) by Ba Dps2 proceeds via an unknown mechanism with an intermediate similar spectrally (A(650 nm)) and kinetically to the maxi-ferritin diferric peroxo complex.     

The so-called Listeria innocua ferritin is a Dps protein. Iron incorporation, detoxification, and DNA protection properties

Listeria innocua Dps (DNA binding protein from starved cells) affords protection to DNA against oxidative damage and can accumulate about 500 iron atoms within its central cavity through a process facilitated by a ferroxidase center. The chemistry of iron binding and oxidation in Listeria Dps (LiDps, formerly described as a ferritin) using H(2)O(2) as oxidant was studied to further define the mechanism of iron deposition inside the protein and the role of LiDps in protecting DNA from oxidative damage. The relatively strong binding of 12 Fe(2+) to the apoprotein (K(D) approximately 0.023 microM) was demonstrated by isothermal titration calorimetry, fluorescence quenching, and pH stat experiments. Hydrogen peroxide was found to be a more efficient oxidant for the protein-bound Fe(2+) than O(2). Iron(II) oxidation by H(2)O(2) occurs with a stoichiometry of 2 Fe(2+)/H(2)O(2) in both the protein-based ferroxidation and subsequent mineralization reactions, indicating complete reduction of H(2)O(2) to H(2)O. Electron paramagnetic resonance (EPR) spin-trapping experiments demonstrated that LiDps attenuates the production of hydroxyl radical by Fenton chemistry. DNA cleavage assays showed that the protein, while not binding to DNA itself, protects it against the deleterious combination of Fe(2+) and H(2)O(2). The overall process of iron deposition and detoxification by LiDps is described by the following equations. For ferroxidation, Fe(2+) + Dps(Z)--> [(Fe(2+))-Dps](Z+1) + H(+) (Fe(2+) binding) and [(Fe(2+))-Dps](Z+1) + Fe(2+) + H(2)O(2) --> [(Fe(3+))(2)(O)(2)-Dps](Z+1) + 2H(+) (Fe(2+) oxidation/hydrolysis). For mineralization, 2Fe(2+) + H(2)O(2) + 2H(2)O --> 2Fe(O)OH((core)) + 4H(+) (Fe(2+) oxidation/hydrolysis). These reactions occur in place of undesirable odd-electron redox processes that produce hydroxyl radical.     

Scavenging of H2O2 and production of oxygen by horseradish peroxidase

Peroxidases catalyze many reactions, the most common being the utilization of H2O2 to oxidize numerous substrates (peroxidative mode). Peroxidases have also been proposed to produce H2O2 via utilization of NAD(P)H, thus providing oxidant either for the first step of lignification or for the "oxidative burst" associated with plant-pathogen interactions. The current study with horseradish peroxidase characterizes a third type of peroxidase activity that mimics the action of catalase; molecular oxygen is produced at the expense of H2O2 in the absence of other reactants. The oxygen production and H2O2-scavenging activities had temperature coefficients, Q10, of nearly 3 and 2, which is consistent with enzymatic reactions. Both activities were inhibited by autoclaving the enzyme and both activities had fairly broad pH optima in the neutral-to-alkaline region. The apparent Km values for the oxygen production and H2O2-scavenging reactions were near 1.0 mM H2O2. Irreversible inactivation of horseradish peroxidase by exposure to high concentrations of H2O2 coincided with the formation of an absorbance peak at 670 nm. Addition of superoxide dismutase (SOD) to reaction mixtures accelerated the reaction, suggesting that superoxide intermediates were involved. It appears that horseradish peroxidase is capable of using H2O2 both as an oxidant and as a reductant. A model is proposed and the relevance of the mechanism in plant-bacterial systems is discussed.     

CD and MCD spectroscopic studies of the two Dps miniferritin proteins from Bacillus anthracis: role of O2 and H2O2 substrates in reactivity of the diiron catalytic centers

DNA protection during starvation (Dps) proteins are miniferritins found in bacteria and archaea that provide protection from uncontrolled Fe(II)/O radical chemistry; thus the catalytic sites are targets for antibiotics against pathogens, such as anthrax. Ferritin protein cages synthesize ferric oxymineral from Fe(II) and O(2)/H(2)O(2), which accumulates in the large central cavity; for Dps, H(2)O(2) is the more common Fe(II) oxidant contrasting with eukaryotic maxiferritins that often prefer dioxygen. To better understand the differences in the catalytic sites of maxi- versus miniferritins, we used a combination of NIR circular dichroism (CD), magnetic circular dichroism (MCD), and variable-temperature, variable-field MCD (VTVH MCD) to study Fe(II) binding to the catalytic sites of the two Bacillus anthracis miniferritins: one in which two Fe(II) react with O(2) exclusively (Dps1) and a second in which both O(2) or H(2)O(2) can react with two Fe(II) (Dps2). Both result in the formation of iron oxybiomineral. The data show a single 5- or 6-coordinate Fe(II) in the absence of oxidant; Fe(II) binding to Dps2 is 30× more stable than Dps1; and the lower limit of K(D) for binding a second Fe(II), in the absence of oxidant, is 2-3 orders of magnitude weaker than for the binding of the single Fe(II). The data fit an equilibrium model where binding of oxidant facilitates formation of the catalytic site, in sharp contrast to eukaryotic M-ferritins where the binuclear Fe(II) centers are preformed before binding of O(2). The two different binding sequences illustrate the mechanistic range possible for catalytic sites of the family of ferritins.     

O2 reactions at the six-iron active site (H-cluster) in [FeFe]-hydrogenase

Irreversible inhibition by molecular oxygen (O(2)) complicates the use of [FeFe]-hydrogenases (HydA) for biotechnological hydrogen (H(2)) production. Modification by O(2) of the active site six-iron complex denoted as the H-cluster ([4Fe4S]-2Fe(H)) of HydA1 from the green alga Chlamydomonas reinhardtii was characterized by x-ray absorption spectroscopy at the iron K-edge. In a time-resolved approach, HydA1 protein samples were prepared after increasing O(2) exposure periods at 0 °C. A kinetic analysis of changes in their x-ray absorption near edge structure and extended X-ray absorption fine structure spectra revealed three phases of O(2) reactions. The first phase (τ(1) ≤ 4 s) is characterized by the formation of an increased number of Fe-O,C bonds, elongation of the Fe-Fe distance in the binuclear unit (2Fe(H)), and oxidation of one iron ion. The second phase (τ(2) ≈ 15 s) causes a ～50% decrease of the number of ～2.7-? Fe-Fe distances in the [4Fe4S] subcluster and the oxidation of one more iron ion. The final phase (τ(3) ≤ 1000 s) leads to the disappearance of most Fe-Fe and Fe-S interactions and further iron oxidation. These results favor a reaction sequence, which involves 1) oxygenation at 2Fe(H(+)) leading to the formation of a reactive oxygen species-like superoxide (O(2)(-)), followed by 2) H-cluster inactivation and destabilization due to ROS attack on the [4Fe4S] cluster to convert it into an apparent [3Fe4S](+) unit, leading to 3) complete O(2)-induced degradation of the remainders of the H-cluster. This mechanism suggests that blocking of ROS diffusion paths and/or altering the redox potential of the [4Fe4S] cubane by genetic engineering may yield improved O(2) tolerance in [FeFe]-hydrogenase.     

H2O2 injury in beta thalassemic erythrocytes: protective role of catalase and the prooxidant effects of GSH

Redox-mediated injury is an important pathway in the destruction of beta thalassemic red blood cells (RBC). Because of the autoxidation of the unstable hemoglobin chains and subsequent release of globin free heme and iron, significant amounts of superoxide (O2-) and, more importantly, hydrogen peroxide (H2O2) are generated intracellularly. Hence, catabolism of H2O2 is crucial in preventing cellular injury. Removal of H2O2 is mediated via two primary pathways: GSH-dependent glutathione peroxidase or catalase. Importantly, both pathways are ultimately dependent on NADPH. In the absence of any exogenous oxidants, model thalassemic RBC demonstrated significantly decreased GSH levels (P < 0.001 at 20 h). Perhaps of greater pathophysiologic importance, however, was the finding that the model thalassemic RBC exhibited significantly (P < 0.001) decreased catalase activity. Following 20 h incubation at 37 degrees C only 61.5 +/- 2.9% of the initial catalase activity remained in the alpha-hemoglobin chain-loaded cells versus 104.6 +/- 4.5 and 108.2 +/- 3.2% in the control and control-resealed cells, respectively. The mechanism underlying the loss of both catalase activity and GSH appears to be the same in that both catabolic pathways require adequate NADPH levels. As shown in this study, model beta thalassemic cells are unable to maintain a normal ( approximately 1.0) NADPH/NADP(total) ratio and, after 20 h, the model beta thalassemic cells have a significantly (P < 0.001) lower ratio ( approximately 0.5) which is quite similar to a G6PD-deficient RBC. In support of these findings, direct inactivation of catalase gives rise to significantly increased oxidant damage. In contrast, GSH depletion is not closely associated with oxidant sensitivity. Indeed, the consumption of GSH noted in the thalassemic RBC may be via a prooxidant pathway as augmentation of cellular GSH levels actually enhances alpha-hemoglobin chain-mediated injury.     

Chelated iron-catalyzed OH. formation from paraquat radicals and H2O2: mechanism of formate oxidation

Traces of iron, when complexed with either EDTA or diethylenetriaminepentaacetic acid (DTPA), catalyze an OH.-producing reaction between H2O2 and paraquat radical (PQ+.): H2O2 + PQ+.----PQ++ + OH. + OH-.[1]. Kinetic studies show that oxidation of formate induced by this reaction occurs by a Fenton-type mechanism, analagous to that assumed in the metal-catalyzed Haber-Weiss reaction, in which the rate determining step is H2O2 + Fe2+ (chelator)----Fe3+(chelator) + OH. + OH-,[7]; with k7 = 7 X 10(3) M-1 s-1 for EDTA and 8 X 10(2) M-1 s-1 for DTPA at pH 7.4. PQ+. rapidly reduces both Fe3+ (EDTA) and Fe3+ (DTPA), and hence allows both agents to catalyze [1] with comparable efficiency, in contrast to the much lower efficiency reported for the latter as a catalyst for the Haber-Weiss reaction. The catalytic properties of these chelating agents is attributed to their lowering of E0 (Fe3+/Fe2+) by 0.65 V, thus making [7] thermodynamically possible at pH 7. Approximately 2.5% of the OH. produced is consumed by internal or "cage" reactions, which decompose the chelator and produce CO2; however, the majority (97%) diffuses into the bulk solution and participates in competitive reactions with OH. scavengers.     

Reactions of peroxyl radicals with Fe(H(2)O)(6)(2+)

The reactions of RO(2)* radicals with Fe(H(2)O)(6)(2+) were studied, R[double bond]H; CH(3); CH(2)COOH; CH(2)CN; CH(2)C(CH(3))(2)OH; CH(2)OH; CHCl(2)/CCl(3). All these processes involve the following reactions: Fe(H(2)O)(6)(2+)+RO(2)*<==>(H(2)O)(5)Fe(III)[bond]OOR(2+) K(1) approximately 250 M(-1); (H(2)O)(5)Fe(III)[bond]OOR(2+)+H(3)O(+)/H(2)O-->Fe(H(2)O)(6)(3+)+ROOH+H(2)O/OH(-); (H(2)O)(5)Fe(III)[bond]OOR(2+)+2Fe(H(2)O)(6)(2+)-->3Fe(H(2)O)(6)(3+)+ROH; 2 RO(2)*-->Products; RO(2)*+(H(2)O)(5)Fe(III)[bond]OOR(2+)-->Fe(H(2)O)(6)(2+)+products. The values of k(1) and k(3) [reaction is clearly not an elementary reaction] approach the ligand exchange rate of Fe(H(2)O)(6)(2+), i.e. these reactions follow an inner sphere mechanism and the rate determining step is the ligand exchange step. The rate of reaction is several orders of magnitude faster than that of the Fenton reaction. Surprisingly enough the K(1) values are nearly independent of the redox potential of the radical and are considerably higher than calculated from the relevant redox potentials. These results indicate that the ROO(-) ligands considerably stabilise the Fe(III) complex, this stabilisation is smaller for radicals with electron withdrawing groups which raise the redox potential of the radical but decrease the basicity of the ROO(-) ligands, two effects which seem to nearly cancel each other. Finally, the results clearly indicate that reaction (5) is relatively fast and affects the nature of the final products. The contribution of these reactions to oxidation processes involving 'Fenton-like' processes is discussed.     

Anaerobic iron deposition into horse spleen, recombinant human heavy and light and bacteria ferritins by large oxidants

Large-molecule oxidants oxidize Fe(II) to form Fe(III) cores in the interior of ferritins at rates comparable to or faster than the iron deposition reaction using O(2) as oxidant. Iron deposition into horse spleen ferritin (HoSF) occurs using ferricyanide ion, 2,6-dichlorophenol-indophenol, and several redox proteins: cytochrome c, stellacyanin, and ceruloplasmin. Cytochrome c also loads iron into recombinant human H-chain (rHF), human L-chain (rLF), and A. vinelandii bacterioferritin (AvBF). The enzymatic activities of ferritins were monitored anaerobically using stopped-flow kinetic spectrophotometry. The reactions exhibit saturation kinetics with respect to the large oxidant concentrations, giving apparent Michaelis constants for cytochrome c as oxidant: K(m)=39.6 microM for HoSF and 6.9 microM for AvBF. Comparison of the kinetic parameters with that of iron deposition by O(2) shows that large oxidants load iron into HoSF and AvBF more effectively than O(2) and may use a mechanism different than the ferroxidase center. Large oxidants did not deposit iron as efficiently with rHF and rLF. The results suggest that the heme groups in AvBF and the protein redox centers present in heteropolymers may assist in anaerobic iron deposition by large oxidants. The physiological relevance of iron deposition by large molecules, including protein oxidants is discussed.     

Copper,zinc superoxide dismutase and H2O2. Effects of bicarbonate on inactivation and oxidations of NADPH and urate,and on consumption of H2O2

Copper,zinc superoxide dismutase (Cu,Zn-SOD) catalyzes the HCO(3)(-)-dependent oxidation of diverse substrates. The mechanism of these oxidations involves the generation of a strong oxidant, derived from H(2)O(2), at the active site copper. This bound oxidant then oxidizes HCO(3)(-) to a strong and diffusible oxidant, presumably the carbonate anion radical that leaves the active site and then oxidizes the diverse substrates. Cu,Zn-SOD is also subject to inactivation by H(2)O(2). It is now demonstrated that the rates of HCO(3)(-)-dependent oxidations of NADPH and urate exceed the rate of inactivation of the enzyme by approximately 100-fold. Cu,Zn-SOD is also seen to catalyze a HCO(3)(-)-dependent consumption of the H(2)O(2) and that HCO(3)(-) does not protect Cu,Zn-SOD against inactivation by H(2)O(2). A scheme of reactions is offered in explanation of these observations.     

Monosaccharide-H2O2 reactions as a source of glycolate and their stimulation by hydroxyl radicals

An analysis of the H(2)O(2)-induced breakdown and transformation of different keto-monosaccharides at physiological concentrations reveals that glycolate and other short-chained carbohydrates and organic acids are produced. Depletion of monosaccharides and glycolate synthesis occurs at increased rates as the length of the carbohydrate chain is decreased, and is significantly increased in the presence of trace amounts of Fe(2+) ions (10 microM). Rates of monosaccharide depletion (initial concentration of 3 mM) observed were up to 1.55 mmol h(-1) in the case of fructose, and 2.59 mmol h(-1) in the case of dihydroxyacetone, depending upon pH, H(2)O(2) concentration, temperature and the presence or absence of catalytic amounts of Fe(2+). Glycolate was produced by dihydroxyacetone cleavage at rates up to 0.45 mmol h(-1) in the absence, and up to 1.88 mmol h(-1) in the presence of Fe(2+) ions (pH 8). Besides glycolate, other sugars (ribose, glyceraldehyde, glucose), glucitol (sorbitol) and organic acids (formic and 2-oxogluconic acid) were produced in such H(2)O(2)-induced reactions with fructose or dihydroxyacetone. EPR measurements demonstrated the participation of the OH radical, especially at higher pH. Presence of metal ions at higher pH values, resulting in increased glycolate synthesis, was accompanied by enhanced hydroxyl radical generation. Observed changes in intensity of DEPMPO-OH signals recorded from dihydroxyacetone and fructose reactions demonstrate a strong correlation with changes in glycolate yield, suggesting that OH radical formation enhances glycolate synthesis. The results presented suggest that different mechanisms are responsible for the cleavage or other reactions (isomerisation, auto- or free-radical-mediated oxidation) of keto-monosaccharides depending of experimental conditions.     

Multiple pathways for mineral core formation in mammalian apoferritin. The role of hydrogen peroxide

Human ferritins sequester and store iron as a stable FeOOH((s)) mineral core within a protein shell assembled from 24 subunits of two types, H and L. Core mineralization in recombinant H- and L-subunit homopolymer and heteropolymer ferritins and several site-directed H-subunit variants was investigated to determine the iron oxidation/hydrolysis chemistry as a function of iron flux into the protein. Stopped-flow absorption spectrometry, UV spectrometry, and electrode oximetry revealed that the mineral core forms by at least three pathways, not two as previously thought. They correspond to the ferroxidase, mineral surface, and the Fe(II) + H2O2 detoxification reactions, respectively: [see reactions]. The H-subunit catalyzed ferroxidase reaction 1 occurs at all levels of iron loading of the protein but decreases with increasing iron added (48-800 Fe(II)/protein). Reaction 2 is the dominant reaction at 800 Fe(II)/protein, whereas reaction 3 occurs largely at intermediate iron loadings of 100-500 Fe(II)/protein. Some of the H2O2 produced in reaction 1 is consumed in the detoxification reaction 3; the 2/1 Fe(II)/H2O2 stoichiometry of reaction 3 minimizes hydroxyl radical production during mineralization. Human L-chain ferritin and H-chain variants lacking functional nucleation and/or ferroxidase sites deposit their iron largely through the mineral surface reaction 2. H2O2 is shown to be an intermediate product of dioxygen reduction in L-chain as well as in H-chain and H-chain variant ferritins.     

Effect of renal medullary H2O2 on salt-induced hypertension and renal injury

Dahl salt-sensitive (SS) and consomic, salt-resistant SS-13(BN) rats possess substantial differences in blood pressure salt-sensitivity even with highly similar genetic backgrounds. The present study examined whether increased oxidative stress, particularly H2O2, in the renal medulla of SS rats contributes to these differences. Blood pressure was measured using femoral arterial catheters in three groups of rats: 1) 12-wk-old SS and consomic SS-13(BN) rats fed a 0.4% NaCl diet, 2) SS rats fed a 4% NaCl diet and chronically infused with saline or catalase (6.9 microg x kg(-1) x min(-1)) directly into the renal medulla, and 3) SS-13(BN) fed high salt (4%) and infused with saline or H2O2 (347 nmol x kg(-1) x min(-1)) into the renal medullary interstitium. After chronic blood pressure measurements, renal medullary interstitial H2O2 concentration ([H2O2]) was collected by microdialysis and analyzed with Amplex red. Blood pressure and [H2O2] were both significantly higher in SS (126 +/- 3 mmHg and 145 +/- 17 nM, respectively) vs. SS-13(BN) rats (116 +/- 2 mmHg and 56 +/- 14 nM) fed a 0.4% diet. Renal interstitial catalase infusion significantly decreased [H2O2] (96 +/- 41 vs. 297 +/- 52 nM) and attenuated the hypertension (146 +/- 2 mmHg catalase vs. 163 +/- 4 mmHg saline) in SS rats after 5 days of high salt (4%). H2O2 infused into the renal medulla of consomic SS-13(BN) fed high salt (4%) for 7 days accentuated the salt sensitivity (145 +/- 2 mmHg H2O2 vs. 134 +/- 1 mmHg saline). [H2O2] was also increased in the treated group (83 +/- 1 nM H2O2 vs. 44 +/- 9 nM saline). These data show that medullary production of H2O2 may contribute to salt-induced hypertension in SS rats and that chromosome 13 of the Brown Norway contains gene(s) that protect against renal medullary oxidant stress.     

Direct voltammetry and electrocatalytic properties of catalase incorporated in polyacrylamide hydrogel films

The direct voltammetry and electrocatalytic properties of catalase (Cat) in polyacrylamide (PAM) hydrogel films cast on pyrolytic graphite (PG) electrodes were investigated. Cat-PAM film electrodes showed a pair of well-defined and nearly reversible cyclic voltammetry peaks for Cat Fe(III)/Fe(II) redox couples at approximately -0.46 V vs. SCE in pH 7.0 buffers. The electron transfer between catalase and PG electrodes was greatly facilitated in the microenvironment of PAM films. The apparent heterogeneous electron transfer rate constant (k(s)) and formal potential (E degrees ') were estimated by fitting square wave voltammograms with non-linear regression analysis. The formal potential of Cat Fe(III)/Fe(II) couples in PAM films had a linear relationship with pH between pH 4.0 and 9.0 with a slope of -56 mV pH(-1), suggesting that one proton is coupled with single-electron transfer for each heme group of catalase in the electrode reaction. UV-Vis absorption spectroscopy demonstrated that catalase retained a near native conformation in PAM films at medium pH. The embedded catalase in PAM films showed the electrocatalytic activity toward dioxygen and hydrogen peroxide. Possible mechanism of catalytic reduction of H(2)O(2) at Cat-PAM film electrodes was proposed.     

Ferritin-catalyzed consumption of hydrogen peroxide by amine buffers causes the variable Fe2+ to O2 stoichiometry of iron deposition in horse spleen ferritin

Ferritin catalyzes the oxidation of Fe2+ by O2 to form a reconstituted Fe3+ oxy-hydroxide mineral core, but extensive studies have shown that the Fe2+ to O2 stoichiometry changes with experimental conditions. At Fe2+ to horse spleen ferritin (HoSF) ratios greater than 200, an upper limit of Fe2+ to O2 of 4 is typically measured, indicating O2 is reduced to 2H2O. In contrast, a lower limit of Fe2+ to O2 of approximately 2 is measured at low Fe2+ to HoSF ratios, implicating H2O2 as a product of Fe2+ deposition. Stoichiometric amounts of H2O2 have not been measured, and H2O2 is proposed to react with an unknown system component. Evidence is presented that identifies this component as amine buffers, including 3-N-morpholinopropanesulfonic acid (MOPS), which is widely used in ferritin studies. In the presence of non-amine buffers, the Fe2+ to O2 stoichiometry was approximately 4.0, but at high concentrations of amine buffers (0.10 M) the Fe2+ to O2 stoichiometry is approximately 2.5 for iron loadings of eight to 30 Fe2+ per HoSF. Decreasing the concentration of amine buffer to zero resulted in an Fe2+ to O2 stoichiometry of approximately 4. Direct evidence for amine buffer modification during Fe2+ deposition was obtained by comparing authentic and modified buffers using mass spectrometry, NMR, and thin layer chromatography. Tris(hydroxymethyl)aminomethane, MOPS, and N-methylmorpholine (a MOPS analog) were all rapidly chemically modified during Fe2+ deposition to form N-oxides. Under identical conditions no modification was detected when amine buffer, H2O2, and O2 were combined with Fe2+ or ferritin separately. Thus, a short-lived ferritin intermediate is required for buffer modification by H2O2. Variation of the Fe2+ to O2 stoichiometry versus the Fe2+ to HoSF ratio and the amine buffer concentration are consistent with buffer modification.     

Fe2+ binding to apo and holo mammalian ferritin

The binding of Fe2+ to both apo and holo mammalian ferritin has been investigated under anaerobic conditions as a function of pH. In the pH range 6.0-7.5, 8.0 +/- 0.5 Fe2+ ions bind to each apoferritin molecule, but above pH 7.5, a pH-dependent Fe2+ binding profile is observed with up to 80 Fe2+ ions binding at pH 10.0. This Fe2+ binding is reversible and is accompanied by up to two H+ being released per Fe2+ bound at pH 10.0. The Fe2+ binding to apoferritin probably occurs in the 3-fold channels. A much larger and more complex pH-dependent Fe2+ binding stoichiometry was observed for holoferritin with up to 300 Fe2+ ions binding at pH 10.0. This pH-dependent Fe2+ binding was interpreted as Fe2+ interaction at the FeOOH mineral surface with displacement of H+ from -OH or phosphate surface groups by the incoming Fe2+ ions. Mossbauer spectroscopic measurements using 57Fe-labeled Fe2+ under anaerobic conditions showed that 57Fe2+ binding to holoferritin was accompanied by electron transfer to the core, yielding 57Fe3+, presumably bound to the mineral surface. Removal of added iron by Fe2+-specific chelating agents yielded 57Fe2+, demonstrating the reversibility of this electron-transfer process. The Fe2+ bound to apo- and holoferritin is readily converted to Fe3+ by exposure to O2 and strongly retained by the respective ferritin species.