Redox processes of sulfur and manganese in a constructed wetland

Constructed wetlands are very popular in terms of wastewater treatment today. Formation of redox potential gradients inside such a system strongly influences the wastewater-treatment efficiency. Individual oxidation forms of sulfur, and dissolved and precipitated manganese forms were determined in the vegetation bed of the constructed wetland. The aim of the speciation analysis was to contribute to the characterization of its redox properties. Sulfur was mostly oxidized at the inflow. The concentration of sulfates at the inflow varied from 25 to 55 mg/l, while concentration of sulfides was always lower than 6.0 mg/l and mostly even lower than 1.0 mg/l. However, sulfates were reduced during the pretreatment and the wastewater flow through the vegetation bed. The concentration of total manganese varied from 0.2 to 0.8 mg/l. Approximately 60% of Mn was precipitated at the inflow. The content of precipitated Mn forms declined to ca. 40-50% at the inflow zone, this content was almost constant across the vegetation bed to the outflow when water was sampled from 60-cm depth. However, the content of precipitated Mn forms increased to ca. 74% for samples from 20-cm depth. With respect to the aeration of the system, manganese can be precipitated as MnO2 in these samples.     

Surface flow constructed wetland for heavy oil-produced water treatment

Heavy oil-produced water from China' Liaohe Oilfield was purified in a surface flow constructed wetland (SFCW) during a 3-yr field experiment. Treatment showed high mean removal efficiencies of 80%, 93%, 88% and 86% for COD, oil, BOD and TKN, respectively for reed bed #1 and 71%, 92%, 77%, and 81% for COD, oil, BOD and TKN, respectively for reed bed #2. The results also showed that in the third year of the system's operation, the oil-produced water had mainly positive impacts on the reed's health parameters. Thus, reed can be used as a feasible wetland macrophyte for treating such wastewater, and this SFCW system can operate for a long time.     

Suboxic deposition of ferric iron by bacteria in opposing gradients of Fe(II) and oxygen at circumneutral pH

The influence of lithotrophic Fe(II)-oxidizing bacteria on patterns of ferric oxide deposition in opposing gradients of Fe(II) and O(2) was examined at submillimeter resolution by use of an O(2) microelectrode and diffusion microprobes for iron. In cultures inoculated with lithotrophic Fe(II)-oxidizing bacteria, the majority of Fe(III) deposition occurred below the depth of O(2) penetration. In contrast, Fe(III) deposition in abiotic control cultures occurred entirely within the aerobic zone. The diffusion microprobes revealed the formation of soluble or colloidal Fe(III) compounds during biological Fe(II) oxidation. The presence of mobile Fe(III) in diffusion probes from live cultures was verified by washing the probes in anoxic water, which removed ca. 70% of the Fe(III) content of probes from live cultures but did not alter the Fe(III) content of probes from abiotic controls. Measurements of the amount of Fe(III) oxide deposited in the medium versus the probes indicated that ca. 90% of the Fe(III) deposited in live cultures was formed biologically. Our findings show that bacterial Fe(II) oxidation is likely to generate reactive Fe(III) compounds that can be immediately available for use as electron acceptors for anaerobic respiration and that biological Fe(II) oxidation may thereby promote rapid microscale Fe redox cycling at aerobic-anaerobic interfaces.     

Suboxic Deposition of Ferric Iron by Bacteria in Opposing Gradients of Fe(II) and Oxygen at Circumneutral pH

The influence of lithotrophic Fe(II)-oxidizing bacteria on patterns of ferric oxide deposition in opposing gradients of Fe(II) and O₂ was examined at submillimeter resolution by use of an O₂ microelectrode and diffusion microprobes for iron. In cultures inoculated with lithotrophic Fe(II)-oxidizing bacteria, the majority of Fe(III) deposition occurred below the depth of O₂ penetration. In contrast, Fe(III) deposition in abiotic control cultures occurred entirely within the aerobic zone. The diffusion microprobes revealed the formation of soluble or colloidal Fe(III) compounds during biological Fe(II) oxidation. The presence of mobile Fe(III) in diffusion probes from live cultures was verified by washing the probes in anoxic water, which removed ca. 70% of the Fe(III) content of probes from live cultures but did not alter the Fe(III) content of probes from abiotic controls. Measurements of the amount of Fe(III) oxide deposited in the medium versus the probes indicated that ca. 90% of the Fe(III) deposited in live cultures was formed biologically. Our findings show that bacterial Fe(II) oxidation is likely to generate reactive Fe(III) compounds that can be immediately available for use as electron acceptors for anaerobic respiration and that biological Fe(II) oxidation may thereby promote rapid microscale Fe redox cycling at aerobic-anaerobic interfaces.     

Further reading in geomicrobiology

In the wetland rhizosphere, high densities of lithotrophic Fe(II)-oxidizing bacteria (FeOB) and a favorable environment (i.e., high Fe(II) availability and microaerobic conditions) suggest that these organisms are actively contributing to the formation of Fe plaque on plant roots. We manipulated the presence/absence of an Fe(II)-oxidizing bacterium (Sideroxydans paludicola, strain BrT) in axenic hydroponic microcosms containing the roots of intact Juncus effusus (soft rush) plants to determine if FeOB affected total rates of rhizosphere Fe(II) oxidation and Fe plaque accumulation. Our experimental data highlight the importance of both FeOB and plants in influencing short-term rates of rhizosphere Fe oxidation. Over time scales ca. 1 wk, the FeOB increased Fe(II) oxidation rates by 1.3 to 1.7 times relative to FeOB-free microcosms. Across multiple experimental trials, Fe(II) oxidation rates were significantly correlated with root biomass, reflecting the importance of radial O ₂ loss in supporting rhizosphere Fe(II) oxidation. Rates of root Fe(III) plaque accumulation (time scales: 3 to 6 wk) were ～ 70 to 83% lower than expected based on the short-term Fe(II) oxidation rates and were unaffected by the presence/absence of FeOB. Decreasing rates of Fe(II) oxidation and Fe(III) plaque accumulation with increasing time scales indicate changes in rates of Fe(II) diffusion and radial O ₂ loss, shifts in the location of Fe oxide accumulation, or temporal changes in the microbial community within the microcosms. The microcosms used herein replicated many of the environmental characteristics of wetland systems and allowed us to demonstrate that FeOB can stimulate rates of Fe(II) oxidation in the wetland rhizosphere, a finding that has implications for the biogeochemical cycling of carbon, metals, and nutrients in wetland ecosystems.     

Oxygen Dependency of Neutrophilic Fe(II) Oxidation by Leptothrix Differs from Abiotic Reaction

Neutrophilic Fe(II) oxidizing microorganisms are found in many natural environments. It has been hypothesized that, at low oxygen concentrations, microbial iron oxidation is favored over abiotic oxidation. Here, we compare the kinetics of abiotic Fe(II) oxidation to oxidation in the presence of the bacterium Leptothrix cholodnii Appels isolated from a wetland sediment. Rates of Fe(II) oxidation were determined in batch experiments at 20°C, pH 7 and oxygen concentrations between 3 and 120 μmol/l. The reaction progress in experiments with and without cells exhibited two distinct phases. During the initial phase, the oxygen dependency of microbial Fe(II) oxidation followed a Michaelis-Menten rate expression (K_M = 24.5 ± 10 μmol O₂/l, v_max = 1.8 ± 0.2 μmol Fe(II)/(l min) for 10⁸ cells/ml). In contrast, abiotic rates increased linearly with increasing oxygen concentrations. At similar oxygen concentrations, initial Fe(II) oxidation rates were faster in the experiments with bacteria. During the second phase, the accumulated iron oxides catalyzed further oxidative iron precipitation in both abiotic and microbial reaction systems. That is, abiotic oxidation also dominated the reaction progress in the presence of bacteria. In fact, in some experiments with bacteria, iron oxidation during the second phase proceeded slower than in the absence of bacteria, possibly due to an inhibitory effect of extracellular polymeric substances on the growth of Fe(III) oxides. Thus, our results suggest that the competitive advantage of microbial iron oxidation in low oxygen environments may be limited by the autocatalytic nature of abiotic Fe(III) oxide precipitation, unless the accumulation of Fe(III) oxides is prevented, for example, through a close coupling of Fe(II) oxidation and Fe(III) reduction.     

Removal of Anionic Surfactants from Wastewater Using a Constructed Wetland

Removal of anionic surfactants from municipal wastewater using a constructed wetland with a horizontal subsurface flow was studied in 2007 and 2008. Extraction spectrophotometry with methylene blue served to determine the analyte concentrations in individual samples. The anionic surfactant‐removal efficiency depended on actual conditions, mostly the treated water flow intensity, its temperature, and a redox‐potential gradient in the longitudinal profile of the wetland bed. It increased with decreasing inflow and increasing temperature. The average efficiency was 83.7% in 2007 and 81.7% in 2008; however, values higher than 85% were often determined during the summer period. On the other hand, the efficiencies were usually lower than 80% in winter, especially in periods with intensive precipitation and inflows. The average concentration of anionic surfactants in water taken at the outflow was lower than 0.65 mg/l (expressed as sodium dodecyl sulfate). The most significant fraction of anionic surfactants (almost 50%) was degraded at the beginning (1 m from the inflow zone) of the wetland bed. The rhizosphere aeration via the vegetation roots strongly supported the anionic‐surfactant removal.     

Physicochemical Conditioning of Dredged Heavy Metal‐Polluted Sediment in Suspension

The remediation of heavy metal‐polluted aquatic sediment by solid‐bed bioleaching requires a material well permeable to air and water. Freshly dredged sediment is nearly impermeable and needs previous conditioning to make it suitable for solid‐bed leaching. This conditioning – in practice carried out by planting sediment packages with helophytes – comprises water removal by evapotranspiration, abiotic and microbial oxidation of sediment‐borne reduced compounds, acidification, as well as structural changes improving the sediment permeability. The rate of this process seems to be limited by the transport of oxygen into the sediment bed. For a better understanding of the physicochemical processes occurring during conditioning, sediment oxidation was studied in a stirred suspension to minimize transport limitations. Freshly dredged, silty, anoxic, heavy metal‐polluted sediment from the Weisse Elster River (Germany) was suspended in water and then continuously stirred and aerated at 20 °C. Aerobic conditions appeared within a few hours. The redox potential increased from – 400 to + 220 mV, at first very quickly and later more slowly. Sediment‐borne inorganic sulfur compounds were oxidized to sulfate (S⁰ mainly within two days and sulfide within ten days), which reduced the pH from 7.2 to 5.9. A successive oxidation of FeS to Fe(II) sulfate, the oxidation of Fe(II) to Fe(III) followed by Fe(III) oxyhydrate formation caused the dissolved Fe to sharply increase and thereafter rapidly decrease. Ammonium was completely oxidized in a nitrification process to form nitrate, further decreasing the pH to 5.5. The acidification increased the solubility of Mn, Zn, Mg, Ca, and K. The increase in dissolved Mn rules out any oxidation of Mn(II) to Mn(IV) since Mn(IV) would have been insoluble under the prevailing pH and redox conditions. Sediment oxidation did not proceed in a well‐defined, redox‐potential‐directed order, but individual (partly microbially) oxidation processes superimposed each other. Physicochemical conditioning of suspended sediment was completed after 20 days while conditioning in a solid bed would require months or even years. These different rates result from transport limitations in the solid bed. Sediment conditioning in a solid bed could therefore possibly be accelerated by prior sediment aeration.     

A process-orientated design and performance assessment methodology for passive mine water treatment systems

The aim of this paper is the development of a methodology for assessing the iron removal efficiency of passive mine water treatment settling lagoons and reed beds. Previous work in the design and sizing of coal mine drainage lagoons and wetlands has focussed on the use of standard hydraulic residence times or using the 10 g m⁻² d⁻¹ metric, these criteria have been applied without regard to the fundamental physical and chemical processes controlling iron removal in the system, namely the pH dependent rates of Fe(II) oxidation and physical settling of the particulate Fe(III). In this study field water quality data have been collected from lagoons and reed beds. These data are examined alongside data from the UK Coal Authority database and combined with simple mathematical formulations to provide a framework through which to understand passive treatment schemes from a process-orientated perspective. It is demonstrated that for the sites studied reed beds are more efficient for iron removal. This paper recommends that judgements of iron removal performance should be based on a derived treatment efficiency index (?) and that the modelling approach espoused in this paper should be used when designing passive mine water treatment schemes.     

Iron binding and oxidation kinetics in frataxin CyaY of Escherichia coli

Friedreich's ataxia is associated with a deficiency in frataxin, a conserved mitochondrial protein of unknown function. Here, we investigate the iron binding and oxidation chemistry of Escherichia coli frataxin (CyaY), a homologue of human frataxin, with the aim of better understanding the functional properties of this protein. Anaerobic isothermal titration calorimetry (ITC) demonstrates that at least two ferrous ions bind specifically but relatively weakly per CyaY monomer (K(d) approximately 4 microM). Such weak binding is consistent with the hypothesis that the protein functions as an iron chaperone. The bound Fe(II) is oxidized slowly by O(2). However, oxidation occurs rapidly and completely with H(2)O(2) through a non-enzymatic process with a stoichiometry of two Fe(II)/H(2)O(2), indicating complete reduction of H(2)O(2) to H(2)O. In accord with this stoichiometry, electron paramagnetic resonance (EPR) spin trapping experiments indicate that iron catalyzed production of hydroxyl radical from Fenton chemistry is greatly attenuated in the presence of CyaY. The Fe(III) produced from oxidation of Fe(II) by H(2)O(2) binds to the protein with a stoichiometry of six Fe(III)/CyaY monomer as independently measured by kinetic, UV-visible, fluorescence, iron analysis and pH-stat titrations. However, as many as 25-26 Fe(III)/monomer can bind to the protein, exhibiting UV absorption properties similar to those of hydrolyzed polynuclear Fe(III) species. Analytical ultracentrifugation measurements indicate that a tetramer is formed when Fe(II) is added anaerobically to the protein; multiple protein aggregates are formed upon oxidation of the bound Fe(II). The observed iron oxidation and binding properties of frataxin CyaY may afford the mitochondria protection against iron-induced oxidative damage.     

Iron homeostasis regulates maturation of tomato (climacteric) and capsicum (non-climacteric) fruits

Journal of Plant Biochemistry and Biotechnology - Endogenous iron quickly interchanges its oxidation states (Fe III and Fe II), which modulate production of reactive oxygen species (ROS) and nitric...     

Iron(III) clusters bound to horse spleen apoferritin: an X-ray absorption and M?ssbauer spectroscopy study that shows that iron nuclei can form on the protein

Ferritin is a complex of a hollow, spherical protein and a hydrous, ferric oxide core of less than or equal to 4500 iron atoms inside the apoprotein coat; the apoprotein has multiple (ca. 12) binding sites for monoatomic metal ions, e.g., Fe(II), V(IV), Tb(III), that may be important in the initiation of iron core formation. In an earlier study we observed that the oxidation of Fe(II) vacated some, but not all, of the metal-binding sites, suggesting migration of some Fe during oxidation, possibly to form nucleation clusters; some Fe(III) remained bound to the protein. Preliminary extended X-ray absorbance fine structure (EXAFS) analysis of the same Fe(III)-apoferritin complex showed an environment distinct from ferritin cores, but the data did not allow a test of the Fe cluster hypothesis. In this paper, with improved EXAFS data and with M?ssbauer data on the same complex formed with 57Fe, we clearly show that the Fe(III) in the distinctive environment is polynuclear (Fe atoms with Fe-Fe = 3.5 A and TB = 7 K). Moreover, the arrangement of atoms is such that Fe(III) atoms appear to have both carboxylate-like ligands, presumably from apoferritin, and oxo bridges to the other iron atoms. Thus the protein provides sites not only for initiation but also for nucleation of the iron core. Sites commodious enough and with sufficient conserved carboxylate ligands to accommodate such a nucleus occur inside the protein coat at the subunit dimer interfaces. Such Fe(III)-apoferritin nucleation complexes can be used to study the properties of the several members of the apoferritin family.     

PCR analysis of the presence and location of Mycobacterium avium in a constructed reed bed, with implications for avian tuberculosis control

The potential of reed beds to act as biofilters of pathogenic and environmental mycobacteria was investigated through examination of the fate of mycobacteria in a constructed reed bed filtering effluent from a large captive wildfowl collection. Particular emphasis was placed on the presence and location of Mycobacterium avium – the causal agent of avian tuberculosis (ATB) – in an effort to clarify the potential role of reed beds in the control of this disease. Water, sediment, and stems and roots of common reed ( Phragmites australis ) and greater reedmace ( Typha latifolia ) were taken from 15 locations within the reed bed plus sites upstream and downstream. Samples were analysed for mycobacteria using PCR and specifically for M. avium using nested PCR. Environmental mycobacteria were found throughout the entire reed bed but M. avium was not found downstream of the first vegetation growth. The reed bed was found to effectively remove M. avium from the water through a combination of sedimentation and adsorption onto vegetation stems. The results of this study show that constructed reed beds composed of a settlement lagoon and one or more vegetation beds can act as valuable and ecologically friendly tools in the environmental control of ATB.     

Iron incorporation into apoferritin. The role of apoferritin as a ferroxidase

Apoferritin catalyzes the oxidation of Fe(II) to Fe(III). Ferroxidase activity is assayed and characterized by coupling the oxidation with the binding of Fe(III) to transferrin. The initial rate of Fe(II) oxidation is dependent on apoferritin and initial Fe(II) concentration but independent of transferrin concentration. The ferroxidase activity is inhibited by Zn(II). Ferritins with varying loads of iron have the same ferroxidase activity level. It is suggested that the described oxidation process represents the initial step of iron deposition in apoferritin. Since transferrin can intercept Fe(III) before it is deposited in apoferritin, active sites for Fe(II) oxidation must be on or near the surface of apoferritin. This finding is contrary to the current view of apoferritin-catalyzed oxidation of Fe(II) which places active sites in the channels to the core or inside the central core.     

Geochemistry of iron in the Salton Sea,California

The Salton Sea is a large, saline, closed-basin lake in southern California. The Sea receives agricultural runoff and, to a lesser extent, municipal wastewater that is high in nutrients, salt, and suspended solids. High sulfate concentrations (4× higher than that of the ocean), coupled with warm temperatures and low-redox potentials present during much of the year, result in extensive sulfate reduction and hydrogen sulfide production. Hydrogen sulfide formation may have a dramatic effect on the iron (Fe) geochemistry in the Sea. We hypothesized that the Fe(II)-sulfide minerals should dominate the iron mineralogy of the sediments, and plans to increase hypolimnetic aeration would increase the amount of Fe(III)-oxides, which are strong adsorbers of phosphate. Sequential chemical extractions were used to differentiate iron mineralogy in the lake sediments and suspended solids from the tributary rivers. Iron in the river-borne suspended solids was mainly associated with structural iron within silicate clays (70%) and ferric oxides (30%). The iron in the bottom sediments of the lake was associated with silicate minerals (71% of the total iron in the sediments), framboidal pyrite (10%), greigite (11%), and amorphous FeS (5%). The ferric oxide fraction was <4% of the total iron in these anaerobic sediments. The morphological characteristics of the framboidal pyrite as determined using SEM suggest that it formed within the water column and experiences some changes in local redox conditions, probably associated with alternating summer anoxia and the well-mixed and generally well-aerated conditions found during the winter. The prevalence of Fe(II)-sulfide minerals in the sediments and the lack of Fe(III)-oxide minerals suggest that the classic model of P-retention by Fe(III)-oxides would not be operating in this lake, at least during anoxic summer conditions. Aeration of the hypolimnion could affect the internal loading of P by changing the relative amounts of Fe(II)-sulfides and Fe(III)-oxides at the sediment/water interface. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. Guest editor: S. H. Hurlbert The Salton Sea Centennial Symposium. Proceedings of a Symposium Celebrating a Century of Symbiosis Among Agriculture, Wildlife and People, 1905–2005, held in San Diego, California, USA, March 2005     

Crystal structures of the NO- and CO-bound heme oxygenase from Neisseriae meningitidis. Implications for O2 activation

Heme oxygenases catalyze the oxidation of heme to biliverdin, carbon monoxide, and free iron while playing a critical role in mammalian heme homeostasis. Pathogenic bacteria such as Neisseriae meningitidis also produce heme oxygenase as part of a mechanism to mine host iron. The key step in heme oxidation is the regioselective oxidation of the heme alpha-meso-carbon by an activated Fe(III)-OOH complex. The structures of various diatomic ligands bound to the heme iron can mimic the dioxygen complex and provide important insights on the mechanism of O2 activation. Here we report the crystal structures of N. meningitidis heme oxygenase (nm-HO) in the Fe(II), Fe(II)-CO, and Fe(II)-NO states and compare these to the NO complex of human heme oxygenase-1 (Lad, L., Wang, J., Li, H., Friedman, J., Bhaskar, B., Ortiz de Montellano, P. R., and Poulos, T. L. (2003) J. Mol. Biol. 330, 527-538). Coordination of NO or CO results in a reorientation of Arg-77 that enables Arg-77 to participate in an active site H-bonded network involving a series of water molecules. One of these water molecules directly H-bonds to the Fe(II)-linked ligand and very likely serves as the proton source required for oxygen activation. Although the active site residues differ between nm-HO and human HO-1, the close similarity in the H-bonded water network suggests a common mechanism shared by all heme oxygenases.     

Life at the energetic edge: kinetics of circumneutral iron oxidation by lithotrophic iron-oxidizing bacteria isolated from the wetland-plant rhizosphere

Batch cultures of a lithotrophic Fe(II)-oxidizing bacterium, strain BrT, isolated from the rhizosphere of a wetland plant, were grown in bioreactors and used to determine the significance of microbial Fe(II) oxidation at circumneutral pH and to identify abiotic variables that affect the partitioning between microbial oxidation and chemical oxidation. Strain BrT grew only in the presence of an Fe(II) source, with an average doubling time of 25 h. In one set of experiments, Fe(II) oxidation rates were measured before and after the cells were poisoned with sodium azide. These experiments indicated that strain BrT accounted for 18 to 53% of the total iron oxidation, and the average cellular growth yield was 0.70 g of CH2O per mol of Fe(II) oxidized. In a second set of experiments, Fe(II) was constantly added to bioreactors inoculated with live cells, killed cells, or no cells. A statistical model fitted to the experimental data demonstrated that metabolic Fe(II) oxidation accounted for up to 62% of the total oxidation. The total Fe(II) oxidation rates in these experiments were strongly limited by the rate of Fe(II) delivery to the system and were also influenced by O2 and total iron concentrations. Additionally, the model suggested that the microbes inhibited rates of abiotic Fe(II) oxidation, perhaps by binding Fe(II) to bacterial exopolymers. The net effect of strain BrT was to accelerate total oxidation rates by up to 18% compared to rates obtained with cell-free treatments. The results suggest that neutrophilic Fe(II)-oxidizing bacteria may compete for limited O2 in the rhizosphere and therefore influence other wetland biogeochemical cycles.     

Removal of Selected Metals from Wastewater Using a Constructed Wetland

Removal of selected metals from municipal wastewater using a constructed wetland with a horizontal subsurface flow was studied. The objective of the work was to determine the efficiency of Cu, Zn, Ni, Co, Sr, Li, and Rb removal, and to describe the main removal mechanisms. The highest removal efficiencies were attained for zinc and copper (89.8 and 81.5%, respectively). It is apparently due to the precipitation of insoluble sulfides (ZnS, CuS) in the vegetation bed where the sulfate reduction takes place. Significantly lower removal efficiencies (43.9, 27.7, and 21.5%) were observed for Li, Sr, and Rb, respectively. Rather, low removal efficiencies were also attained for Ni and Co (39.8 and 20.9%). However, the concentrations of these metals in treated water were significantly lower compared to Cu and Zn (e.g., 2.8 ± 0.5 and 1.7 ± 0.3 μg/l for Ni at the inflow and outflow from the wetland compared to 27.6 ± 12.0 and 5.1 ± 4.7 μg/l obtained for Cu, respectively). The main perspective of the constructed wetland is the removal of toxic heavy metals forming insoluble compounds depositing in the wetland bed. Metal uptake occurs preferentially in wetland sediments and is closely associated with the chemism of sulfur and iron.     

EPR detection of protein-derived radicals in the reaction of H(2)O(2) with Fe bound in mitochondrial F(1)ATPase.

A severe inactivation is obtained upon the addition of H(2)O(2) to bovine heart F(1)ATPase samples containing Fe(III) in the nucleotide-independent site, and Fe(II) in the ATP-dependent site. EPR spectra at 4.9 K of these samples indicate that H(2)O(2) produces the complete oxidation of Fe(II) to Fe(III) and the concomitant appearance of two protein-derived radical species. The two signals (g = 2.036 and g = 2.007) display a different temperature dependence and saturation behavior. The relaxation properties of the radical at g = 2.036 suggest magnetic interaction with one of the two iron centers. Such events are not observed when H(2)O(2) is added either to native F(1)ATPase containing a high amount of Fe(II) and low amount of Fe(III) or to F(1)ATPase deprived of endogenous Fe and subsequently loaded with only Fe(III) in both sites. It is hypothesized that in F(1)ATPase samples containing both Fe(III) and Fe(II), intramolecular long-range electron transfer may occur from Fe(II) to a high oxidation state species of Fe formed in the nucleotide-independent site upon oxidation of Fe(III) by H(2)O(2).