Characterization of Mn(II) and Mn(III) binding capability of natural siderophores desferrioxamine B and desferricoprogen as well as model hydroxamic acids

Complexes formed between Mn(II) ion and acetohydroxamic acid (HAha), benzohydroxamic acid (HBha), N-methyl-acetohydroxamic acid (HMeAha), DFB model dihydroxamic acids (H₂(3,4-DIHA), H₂(3,3-DIHA), H₂(2,5-DIHA), H₂(2,5-H,H-DIHA), H₂(2,4-DIHA), H₂(2,3-DIHA)) and two trihydroxamate based natural siderophores, desferrioxamine B (H₄DFB) and desferricoprogen (H₃DFC) have been investigated under anaerobic condition (and some of them also under aerobic condition). The pH-potentiometric results showed the formation of well-defined complexes with moderate stability. Monohydroxamic acids not, but all of the dihydroxamic acids and trihydroxamic acids were able to hinder the hydrolysis of the metal ion up to pH ca. 11. Maximum three hydroxamates were found to coordinate to the Mn(II) ion, but presence of water molecule in the inner-sphere was also indicated by the corresponding relaxivity values even in the tris-chelated complexes. Moreover, prototropic exchange processes were found to increase the relaxation rate of the solvent water proton over the value of [Mn_aqua]²⁺ in the protonated Mn(II)-siderophore complexes at physiological pH. The much higher stability of Mn(III)-hydroxamate (especially tris-chelated) complexes compared to the corresponding Mn(II)-containing species results in a significantly decreased formal potential compared to the Mn(III)_aqua/Mn(II)_aqua system. As a result, air oxygen becomes an oxidizing agent for these manganese(II)-hydroxamate complexes above pH 7.5. The oxidation processes, followed by UV-Vis spectrophotometry, were found to be stoichiometric only in the case of the tris-chelated complexes of siderophores, which predominate above pH 9. ESI-MS provided support about the stoichiometry and cyclic-voltammetry was used to determine the stability constants for the tris-chelated complexes, [Mn(HDFB)]⁺ and [MnDFC].     

31P and 17O NMR of Mn(III)-containing acid phosphatase: Evidence for direct metal-phosphate interaction and oxygen exchange from water into inorganic phosphate

The acid phosphatase isolated from sweet potato tubers by us is unique Mn(III)-containing enzyme which hydrolyzes phosphomonoesters and nucleotide phosphates. The present ³¹P and ¹⁷O NMR studies of the Mn(III)-containing acid phosphatase solved two important problems. The broadening of the phosphate ³¹P resonance signal in the 1:1 enzyme-substrate system shows evidence for direct metal-phosphate interaction in the Mn(III)-containing acid phosphatase. In addition, the ¹⁷O NMR evidence for oxygen exchange from water into inorganic phosphate strongly indicates that the Mn(III)-containing acid phosphatase catalyzes an apparent transition state displacement and P-O cleavage as follows: $\text{ROPO}{}_3{}^{\mathrm{=}}\text{+ H}{}^{17}\text{OHROH + H}{}^{17}\text{OPO}{}_3{}^{\mathrm{=}}$.     

Magnetic resonance studies of Mn(II)-, Mn(III)-, and Fe(III)-conalbumin complexes.

Apoconalbumin binds Mn(II) at two sites with association constants of K1 = 7 (+/- 1) X 10(4) and K2 = 0.4 (+/- 0.25) X 10(4) M-1. The binding is tighter in the presence of excess bicarbonate resulting in K1 = 1.8 (+/- 0.2) X 10(5) and K2 = 3 (+/- 2) X 10(4) M-1. The electron paramagnetic resonance spectrum (at both 9 and 35 GHz) of Mn(II) bound at the tight site reveals a rhombic distortion (lambda = E/D approximately equal to 0.25-0.31) in the protein ligand environment of the mental ion. An evaluation of the 1/pT1p, paramagnetic contribution to the longitudinal relaxation rate of solvent protons with Mn(II)-, Mn(III)-, and Fe(III)-derivatives of conalbumin revealed that the mental ion in each site of conalbumin is accessible to one water molecule. For Mn(II)-conalbumin and Mn(III)-conalbumin species, inner coordination sphere protons are rapidly exchanging with the bulk solvent, while slow exchange conditions prevail for Fe(III)-conalbumin.     

Comparison of the SOD-like activity of hexacoordinate Mn(II), Fe(II) and Ni(II) complexes having isoindoline-based ligands

Recently, a series of Fe(II) complexes have been published by our group with 3 N-donor 1,3-bis(2′-Ar-imino)isoindoline ligands containing various Ar-groups (pyridyl, 4-methylpyridyl, thiazolyl, benzimidazolyl and N-methylbenzimidazolyl). The superoxide scavenging activity of the compounds showed correlation with the Fe(III)/Fe(II) redox potentials. Analogous, electroneutral chelate complexes with Mn(II) and Ni(II) in 2:1 ligand:metal composition are reported here. Each Mn(II) complex exhibits one reversible redox wave that is assigned as the Mn(III)/Mn(II) redox transition. The E_1/2 spans a 180 mV range from − 98 (Ar = 3-methylpyridyl) to 82 mV (Ar = thiazolyl) vs. the Fc⁺/Fc depending on the Ar-sidearm. The SOD-like (SOD=superoxide dismutase)activity of all complexes was determined according to the McCord-Fridovich method. The Mn(II) isoindolinates have IC₅₀ values - determined with 50 μM cytochrome c Fe(III) - that range from (3.22 ± 0.39) × 10^− 6 (Ar = benzimidazolyl) to (10.80 ± 0.54) × 10^− 6 M (Ar = N-methylbenzimidazolyl). In contrast with the Fe(II) complexes, the IC₅₀ concentrations show no significant dependence on the E_1/2 values in this narrow potential range emphasizing that the redox potential is not the governing factor in the Mn(II)-containing scavengers. The analogous Ni(II) compounds show no redox transitions in the thermodynamically relevant potential range (− 0.40 to 0.65 V vs. SCE) and accordingly, their superoxide scavenging activity (if any) is below the detection level.     

Dissimilatory Fe(III) Reduction by the Marine Microorganism Desulfuromonas acetoxidans

The ability of the marine microorganism Desulfuromonas acetoxidans to reduce Fe(III) was investigated because of its close phylogenetic relationship with the freshwater dissimilatory Fe(III) reducer Geobacter metallireducens. Washed cell suspensions of the type strain of D. acetoxidans reduced soluble Fe(III)-citrate and Fe(III) complexed with nitriloacetic acid. The c-type cytochrome(s) of D. acetoxidans was oxidized by Fe(III)-citrate and Mn(IV)-oxalate, as well as by two electron acceptors known to support growth, colloidal sulfur and malate. D. acetoxidans grew in defined anoxic, bicarbonate-buffered medium with acetate as the sole electron donor and poorly crystalline Fe(III) or Mn(IV) as the sole electron acceptor. Magnetite (Fe₃O₄) and siderite (FeCO₃) were the major end products of Fe(III) reduction, whereas rhodochrosite (MnCO₃) was the end product of Mn(IV) reduction. Ethanol, propanol, pyruvate, and butanol also served as electron donors for Fe(III) reduction. In contrast to D. acetoxidans, G. metallireducens could only grow in freshwater medium and it did not conserve energy to support growth from colloidal S⁰ reduction. D. acetoxidans is the first marine microorganism shown to conserve energy to support growth by coupling the complete oxidation of organic compounds to the reduction of Fe(III) or Mn(IV). Thus, D. acetoxidans provides a model enzymatic mechanism for Fe(III) or Mn(IV) oxidation of organic compounds in marine and estuarine sediments. These findings demonstrate that 16S rRNA phylogenetic analyses can suggest previously unrecognized metabolic capabilities of microorganisms.     

pH Gradient-Induced Heterogeneity of Fe(III)-Reducing Microorganisms in Coal Mining-Associated Lake Sediments

Lakes formed because of coal mining are characterized by low pH and high concentrations of Fe(II) and sulfate. The anoxic sediment is often separated into an upper acidic zone (pH 3; zone I) with large amounts of reactive iron and a deeper slightly acidic zone (pH 5.5; zone III) with smaller amounts of iron. In this study, the impact of pH on the Fe(III)-reducing activities in both of these sediment zones was investigated, and molecular analyses that elucidated the sediment microbial diversity were performed. Fe(II) was formed in zone I and III sediment microcosms at rates that were approximately 710 and 895 nmol cm⁻³ day⁻¹, respectively. A shift to pH 5.3 conditions increased Fe(II) formation in zone I by a factor of 2. A shift to pH 3 conditions inhibited Fe(II) formation in zone III. Clone libraries revealed that the majority of the clones from both zones (approximately 44%) belonged to the Acidobacteria phylum. Since moderately acidophilic Acidobacteria species have the ability to oxidize Fe(II) and since Acidobacterium capsulatum reduced Fe oxides at pHs ranging from 2 to 5, this group appeared to be involved in the cycling of iron. PCR products specific for species related to Acidiphilium revealed that there were higher numbers of phylotypes related to cultured Acidiphilium or Acidisphaera species in zone III than in zone I. From the PCR products obtained for bioleaching-associated bacteria, only one phylotype with a level of similarity to Acidithiobacillus ferrooxidans of 99% was obtained. Using primer sets specific for Geobacteraceae, PCR products were obtained in higher DNA dilutions from zone III than from zone I. Phylogenetic analysis of clone libraries obtained from Fe(III)-reducing enrichment cultures grown at pH 5.5 revealed that the majority of clones were closely related to members of the Betaproteobacteria, primarily species of Thiomonas. Our results demonstrated that the upper acidic sediment was inhabited by acidophiles or moderate acidophiles which can also reduce Fe(III) under slightly acidic conditions. The majority of Fe(III) reducers inhabiting the slightly acidic sediment had only minor capacities to be active under acidic conditions.     

Absence of humic substance reduction by the acidophilic Fe(III)-reducing strain Acidiphilium SJH: implications for its Fe(III) reduction mechanism and for the stimulation of natural organohalogen formation

A vast amount of volatile organohalogens (VOX) has natural origins. Both soils and sediments have been shown to release VOX, which are most likely produced via redox reactions between Fe(III) and quinones in the presence of halide anions, particularly at acidic pH. We tested whether acidophilic Fe(III)-reducers might indirectly stimulate natural VOX formation at acidic pH by providing reactive Fe and quinone species. However, it is unknown whether acidophilic Fe(III)-reducers can reduce humic acids (HA) or fulvic acids (FA). We therefore tested the ability of the acidophilic Fe(III)-reducer Acidiphilium SJH to reduce macromolecular, suspended HA and dissolved FA at pH 3.1–3.3. We found that (i) SJH can neither reduce HA/FA nor the humic model quinone anthraquinone-2,6-disulfonic-acid (AQDS) nor stimulate the formation of FA radicals, (ii) at acidic pH, significantly more electrons are transferred abiotically both from native and reduced FA to dissolved Fe(III) than from native or reduced HA, and (iii) the presence of strain SJH does not stimulate VOX formation. Our results imply that the acidophilic Fe(III)-reducer SJH either uses an enzyme for Fe(III) reduction that can neither be used for HA/FA nor for AQDS reduction or that the location of Fe(III) reduction is inaccessible for these compounds. We further conclude that microorganisms such as strain SJH probably do not indirectly stimulate natural VOX formation at acidic pH via the formation of reactive quinone species.     

Evidence for nonbridged coordination of p-nitrophenyl phosphate to the dinuclear Fe(III)-M(II) center in bovine spleen purple acid phosphatase during enzymatic turnover.

The pH dependence of the catalytic parameters k(cat) and K(M) has been determined for the Fe(III)Fe(II)- and Fe(III)Zn(II)-forms of bovine spleen purple acid phosphatase (BSPAP). The parameter k(cat) was found to be maximal at pH 6.3, and a pK(a) of 5.4-5.5 was obtained for the acidic limb of the k(cat) vs pH profile. Two different EPR spectra were detected for the phosphate complex of the mixed-valent diiron enzyme; their relative amounts depended on the pH, with an apparent pK(a) of 6. The EPR spectra of Fe(III)Fe(II)-BSPAP.PO(4) and Fe(III)Zn(II)-BSPAP.PO(4) at pH 5.0 are similar to those previously reported for Fe(III)Fe(II)-Uf.PO(4) and Fe(III)Zn(II)-Uf.PO(4) complexes at pH 5.0. At higher pH, a new Fe(III)Fe(II)-BSPAP.PO(4) species is formed, with apparent g-values of 1.94, 1.71, and 1.50. The EPR spectrum of Fe(III)Zn(II)-BSPAP does not show significant changes upon addition of phosphate up to 30 mM at pH 6.5, suggesting that phosphate binds only to the spectroscopically silent Zn(II). To determine whether the phosphate complexes were good structural models for the enzyme substrate complexes, these complexes were studied using rapid-freeze EPR and stopped-flow optical spectroscopy. The stopped-flow studies showed the absence of burst kinetics at pH 7.0, which indicates that substrate hydrolysis is rate limiting, rather than phosphate release. The EPR spectrum of Fe(III)Fe(II)-BSPAP.p-NPP is similar, but not identical, to that of the corresponding phosphate complex, both at pH 5 and pH 6.5. We propose that both phosphate and p-NPP bridge the two metal ions at low pH. At higher pH where the enzyme is optimally active, we propose that hydroxide competes with phosphate and p-NPP for coordination to Fe(III) and that both phosphate and p-NPP coordinate only to the divalent metal ion.     

Reduction of Fe(III), Mn(III), and Cu(II) chelates by roots of pea (Pisum sativum L.) or soybean (Glycine max)

Neither the reduction of Fe(III) to Fe(II) by roots nor its induction by Fe-deficiency are unique characteristics of the reductive activities of roots. We show that chelated Mn(III) or chelated Cu(II), as well as chelated Fe(III), may be reduced by Fe-stressed roots of pea (Pisum sativum L.). Deficiency of Fe stimulated the reduction of Fe(III)EDTA about 20-fold, the reduction of Mn(III)CDTA about 11-fold, the reduction of Cu(II)(BPDS)₂ about 5-fold, and the reduction of Fe(III)(CN)₆ by only about 50%. Not only are metals other than Fe reduced as part of the Fe-stress response, but deficiencies of metals other than Fe stimulate the reductive activity of roots. We show that depriving peas or soybeans (Glycine max) of Cu or Zn stimulates the reduction of Fe(III).     

Direct and Fe(II)-Mediated Reduction of Technetium by Fe(III)-Reducing Bacteria

The dissimilatory Fe(III)-reducing bacterium Geobacter sulfurreducens reduced and precipitated Tc(VII) by two mechanisms. Washed cell suspensions coupled the oxidation of hydrogen to enzymatic reduction of Tc(VII) to Tc(IV), leading to the precipitation of TcO₂ at the periphery of the cell. An indirect, Fe(II)-mediated mechanism was also identified. Acetate, although not utilized efficiently as an electron donor for direct cell-mediated reduction of technetium, supported the reduction of Fe(III), and the Fe(II) formed was able to transfer electrons abiotically to Tc(VII). Tc(VII) reduction was comparatively inefficient via this indirect mechanism when soluble Fe(III) citrate was supplied to the cultures but was enhanced in the presence of solid Fe(III) oxide. The rate of Tc(VII) reduction was optimal, however, when Fe(III) oxide reduction was stimulated by the addition of the humic analog and electron shuttle anthaquinone-2,6-disulfonate, leading to the rapid formation of the Fe(II)-bearing mineral magnetite. Under these conditions, Tc(VII) was reduced and precipitated abiotically on the nanocrystals of biogenic magnetite as TcO₂ and was removed from solution to concentrations below the limit of detection by scintillation counting. Cultures of Fe(III)-reducing bacteria enriched from radionuclide-contaminated sediment using Fe(III) oxide as an electron acceptor in the presence of 25 μM Tc(VII) contained a single Geobacter sp. detected by 16S ribosomal DNA analysis and were also able to reduce and precipitate the radionuclide via biogenic magnetite. Fe(III) reduction was stimulated in aquifer material, resulting in the formation of Fe(II)-containing minerals that were able to reduce and precipitate Tc(VII). These results suggest that Fe(III)-reducing bacteria may play an important role in immobilizing technetium in sediments via direct and indirect mechanisms.     

Coexistence of Microaerophilic,Nitrate-Reducing,and Phototrophic Fe(II) Oxidizers and Fe(III) Reducers in Coastal Marine Sediment

Iron is abundant in sediments, where it can be biogeochemically cycled between its divalent and trivalent redox states. The neutrophilic microbiological Fe cycle involves Fe(III)-reducing and three different physiological groups of Fe(II)-oxidizing microorganisms, i.e., microaerophilic, anoxygenic phototrophic, and nitrate-reducing Fe(II) oxidizers. However, it is unknown whether all three groups coexist in one habitat and how they are spatially distributed in relation to gradients of O₂, light, nitrate, and Fe(II). We examined two coastal marine sediments in Aarhus Bay, Denmark, by cultivation and most probable number (MPN) studies for Fe(II) oxidizers and Fe(III) reducers and by quantitative-PCR (qPCR) assays for microaerophilic Fe(II) oxidizers. Our results demonstrate the coexistence of all three metabolic types of Fe(II) oxidizers and Fe(III) reducers. In qPCR, microaerophilic Fe(II) oxidizers (Zetaproteobacteria) were present with up to 3.2 × 10⁶ cells g dry sediment⁻¹. In MPNs, nitrate-reducing Fe(II) oxidizers, anoxygenic phototrophic Fe(II) oxidizers, and Fe(III) reducers reached cell numbers of up to 3.5 × 10⁴, 3.1 × 10², and 4.4 × 10⁴ g dry sediment⁻¹, respectively. O₂ and light penetrated only a few millimeters, but the depth distribution of the different iron metabolizers did not correlate with the profile of O₂, Fe(II), or light. Instead, abundances were homogeneous within the upper 3 cm of the sediment, probably due to wave-induced sediment reworking and bioturbation. In microaerophilic Fe(II)-oxidizing enrichment cultures, strains belonging to the Zetaproteobacteria were identified. Photoferrotrophic enrichments contained strains related to Chlorobium and Rhodobacter; the nitrate-reducing Fe(II) enrichments contained strains related to Hoeflea and Denitromonas. This study shows the coexistence of all three types of Fe(II) oxidizers in two near-shore marine environments and the potential for competition and interrelationships between them.     

Manganese(IV) Oxide Production by Acremonium sp. Strain KR21-2 and Extracellular Mn(II) Oxidase Activity

Ascomycetes that can deposit Mn(III, IV) oxides are widespread in aquatic and soil environments, yet the mechanism(s) involved in Mn oxide deposition remains unclear. A Mn(II)-oxidizing ascomycete, Acremonium sp. strain KR21-2, produced a Mn oxide phase with filamentous nanostructures. X-ray absorption near-edge structure (XANES) spectroscopy showed that the Mn phase was primarily Mn(IV). We purified to homogeneity a laccase-like enzyme with Mn(II) oxidase activity from cultures of strain KR21-2. The purified enzyme oxidized Mn(II) to yield suspended Mn particles; XANES spectra indicated that Mn(II) had been converted to Mn(IV). The pH optimum for Mn(II) oxidation was 7.0, and the apparent half-saturation constant was 0.20 mM. The enzyme oxidized ABTS [2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid)] (pH optimum, 5.5; K_m, 1.2 mM) and contained two copper atoms per molecule. Moreover, the N-terminal amino acid sequence (residues 3 to 25) was 61% identical with the corresponding sequence of an Acremonium polyphenol oxidase and 57% identical with that of a Myrothecium bilirubin oxidase. These results provide the first evidence that a fungal multicopper oxidase can convert Mn(II) to Mn(IV) oxide. The present study reinforces the notion of the contribution of multicopper oxidase to microbially mediated precipitation of Mn oxides and suggests that Acremonium sp. strain KR21-2 is a good model for understanding the oxidation of Mn in diverse ascomycetes.     

Characterization of Fe(III) Reduction by Chlororespiring Anaeromxyobacter dehalogenans

Anaeromyxobacter dehalogenans strain 2CP-C has been shown to grow by coupling the oxidation of acetate to the reduction of ortho-substituted halophenols, oxygen, nitrate, nitrite, or fumarate. In this study, strain 2CP-C was also found to grow by coupling Fe(III) reduction to the oxidation of acetate, making it one of the few isolates capable of growth by both metal reduction and chlororespiration. Doubling times for growth of 9.2 and 10.2 h were determined for Fe(III) and 2-chlorophenol reduction, respectively. These were determined by using the rate of [¹⁴C]acetate uptake into biomass. Fe(III) compounds used by strain 2CP-C include ferric citrate, ferric pyrophosphate, and amorphous ferric oxyhydroxide. The addition of the humic acid analog anthraquinone 2,6-disulfonate (AQDS) increased the reduction rate of amorphous ferric iron oxide, suggesting AQDS was used as an electron shuttle by strain 2CP-C. The addition of chloramphenicol to fumarate-grown cells did not inhibit Fe(III) reduction, indicating that the latter activity is constitutive. In contrast, the addition of chloramphenicol inhibited dechlorination activity, indicating that chlororespiration is inducible. The presence of insoluble Fe(III) oxyhydroxide did not significantly affect dechlorination, whereas the presence of soluble ferric pyrophosphate inhibited dechlorination. With its ability to respire chlorinated organic compounds and metals such as Fe(III), strain 2CP-C is a promising model organism for the study of the interaction of these potentially competing processes in contaminated environments.     

Mn(II) oxidation is the principal function of the extracellular Mn-peroxidase from Phanerochaete chrysosporium

The manganese peroxidase (MnP), from the lignin-degrading fungus Phanerochaete chrysosporium, an H2O2-dependent heme enzyme, oxidizes a variety of organic compounds but only in the presence of Mn(II). The homogeneous enzyme rapidly oxidizes Mn(II) to Mn(III) with a pH optimum of 5.0; the latter was detected by the characteristic spectrum of its lactate complex. In the presence of H2O2 the enzyme oxidizes Mn(II) significantly faster than it oxidizes all other substrates. Addition of 1 M equivalent of H2O2 to the native enzyme in 20 mM Na-succinate, pH 4.5, yields MnP compound II, characterized by a Soret maximum at 416 nm. Subsequent addition of 1 M equivalent of Mn(II) to the compound II form of the enzyme results in its rapid reduction to the native Fe3+ species. Mn(III)-lactate oxidizes all of the compounds which are oxidized by the enzymatic system. The relative rates of oxidation of various substrates by the enzymatic and chemical systems are similar. In addition, when separated from the polymeric dye Poly B by a semipermeable membrane, the enzyme in the presence of Mn(II)-lactate and H2O2 oxidizes the substrate. All of these results indicate that the enzyme oxidizes Mn(II) to Mn(III) and that the Mn(III) complexed to lactate or other alpha-hydroxy acids acts as an obligatory oxidation intermediate in the oxidation of various dyes and lignin model compounds. In the absence of exogenous H2O2, the Mn-peroxidase oxidized NADH to NAD+, generating H2O2 in the process. The H2O2 generated by the oxidation of NADH could be utilized by the enzyme to oxidize a variety of other substrates.     

Microbial Community Analysis and Effects of Fe(II)/Mn(II) Ratio on Denitrification in the Mixotrophic Biological Reactor

In this study, the denitrification performance of the mixotrophic biological reactor was investigated under varying Fe(II)/Mn(II) molar ratio conditions. Results indicate that the optimal nitrate removal ratio occurred at an Fe(II)/Mn(II) molar ratio of 9:1, pH of 7, with an HRT of 10?h. When the reactor was performing under optimal conditions, the nitrate removal reached 100.00% at a rate of 0.116?mmol·L^?1·h^?1. The proportion of oxidized Fe(II) and Mn(II) reached 99.29% and 21.88%, respectively. High-throughput sequencing results show that Pseudomonas was the dominant species in the mixotrophic biological reactor. Furthermore, the relative abundance of Pseudomonas and denitrification performance was significantly influenced by variation in the Fe(II)/Mn(II) molar ratio.     

Reduction of Fe(III), Mn(IV), and Toxic Metals at 100°C by Pyrobaculum islandicum

It has recently been noted that a diversity of hyperthermophilic microorganisms have the ability to reduce Fe(III) with hydrogen as the electron donor, but the reduction of Fe(III) or other metals by these organisms has not been previously examined in detail. When Pyrobaculum islandicum was grown at 100°C in a medium with hydrogen as the electron donor and Fe(III)-citrate as the electron acceptor, the increase in cell numbers of P. islandicum per mole of Fe(III) reduced was found to be ca. 10-fold higher than previously reported. Poorly crystalline Fe(III) oxide could also serve as the electron acceptor for growth on hydrogen. The stoichiometry of hydrogen uptake and Fe(III) oxide reduction was consistent with the oxidation of 1 mol of hydrogen resulting in the reduction of 2 mol of Fe(III). The poorly crystalline Fe(III) oxide was reduced to extracellular magnetite. P. islandicum could not effectively reduce the crystalline Fe(III) oxide minerals goethite and hematite. In addition to using hydrogen as an electron donor for Fe(III) reduction, P. islandicum grew via Fe(III) reduction in media in which peptone and yeast extract served as potential electron donors. The closely related species P. aerophilum grew via Fe(III) reduction in a similar complex medium. Cell suspensions of P. islandicum reduced the following metals with hydrogen as the electron donor: U(VI), Tc(VII), Cr(VI), Co(III), and Mn(IV). The reduction of these metals was dependent upon the presence of cells and hydrogen. The metalloids arsenate and selenate were not reduced. U(VI) was reduced to the insoluble U(IV) mineral uraninite, which was extracellular. Tc(VII) was reduced to insoluble Tc(IV) or Tc(V). Cr(VI) was reduced to the less toxic, less soluble Cr(III). Co(III) was reduced to Co(II). Mn(IV) was reduced to Mn(II) with the formation of manganese carbonate. These results demonstrate that biological reduction may contribute to the speciation of metals in hydrothermal environments and could account for such phenomena as magnetite accumulation and the formation of uranium deposits at ca. 100°C. Reduction of toxic metals with hyperthermophilic microorganisms or their enzymes might be applied to the remediation of metal-contaminated waters or waste streams.     

Shewanella putrefaciens mtrB Encodes an Outer Membrane Protein Required for Fe(III) and Mn(IV) Reduction

Iron and manganese oxides or oxyhydroxides are abundant transition metals, and in aquatic environments they serve as terminal electron acceptors for a large number of bacterial species. The molecular mechanisms of anaerobic metal reduction, however, are not understood. Shewanella putrefaciens is a facultative anaerobe that uses Fe(III) and Mn(IV) as terminal electron acceptors during anaerobic respiration. Transposon mutagenesis was used to generate mutants of S. putrefaciens, and one such mutant, SR-21, was analyzed in detail. Growth and enzyme assays indicated that the mutation in SR-21 resulted in loss of Fe(III) and Mn(IV) reduction but did not affect its ability to reduce other electron acceptors used by the wild type. This deficiency was due to Tn5 inactivation of an open reading frame (ORF) designated mtrB. mtrB encodes a protein of 679 amino acids and contains a signal sequence characteristic of secreted proteins. Analysis of membrane fractions of the mutant, SR-21, and wild-type cells indicated that MtrB is located on the outer membrane of S. putrefaciens. A 5.2-kb DNA fragment that contains mtrB was isolated and completely sequenced. A second ORF, designated mtrA, was found directly upstream of mtrB. The two ORFs appear to be arranged in an operon. mtrA encodes a putative 10-heme c-type cytochrome of 333 amino acids. The N-terminal sequence of MtrA contains a potential signal sequence for secretion across the cell membrane. The amino acid sequence of MtrA exhibited 34% identity to NrfB from Escherichia coli, which is involved in formate-dependent nitrite reduction. To our knowledge, this is the first report of genes encoding proteins involved in metal reduction.     

Multicopper oxidase involvement in both Mn(II) and Mn(III) oxidation during bacterial formation of MnO2

Global cycling of environmental manganese requires catalysis by bacteria and fungi for MnO₂ formation, since abiotic Mn(II) oxidation is slow under ambient conditions. Genetic evidence from several bacteria indicates that multicopper oxidases (MCOs) are required for MnO₂ formation. However, MCOs catalyze one-electron oxidations, whereas the conversion of Mn(II) to MnO₂ is a two-electron process. Trapping experiments with pyrophosphate (PP), a Mn(III) chelator, have demonstrated that Mn(III) is an intermediate in Mn(II) oxidation when mediated by exosporium from the Mn-oxidizing bacterium Bacillus SG-1. The reaction of Mn(II) depends on O₂ and is inhibited by azide, consistent with MCO catalysis. We show that the subsequent conversion of Mn(III) to MnO₂ also depends on O₂ and is inhibited by azide. Thus, both oxidation steps appear to be MCO-mediated, likely by the same enzyme, which is indicated by genetic evidence to be the MnxG gene product. We propose a model of how the manganese oxidase active site may be organized to couple successive electron transfers to the formation of polynuclear Mn(IV) complexes as precursors to MnO₂ formation.     

Regulation of Dissimilatory Fe(III) Reduction Activity in Shewanella putrefaciens

Under anaerobic conditions, Shewanella putrefaciens is capable of respiratory-chain-linked, high-rate dissimilatory iron reduction via both a constitutive and inducible Fe(III)-reducing system. In the presence of low levels of dissolved oxygen, however, iron reduction by this microorganism is extremely slow. Fe(II)-trapping experiments in which Fe(III) and O₂ were presented simultaneously to batch cultures of S. putrefaciens indicated that autoxidation of Fe(II) was not responsible for the absence of Fe(III) reduction. Inhibition of cytochrome oxidase with CN⁻ resulted in a high rate of Fe(III) reduction in the presence of dissolved O₂, which suggested that respiratory control mechanisms did not involve inhibition of Fe(III) reductase activities or Fe(III) transport by molecular oxygen. Decreasing the intracellular ATP concentrations by using an uncoupler, 2,4-dinitrophenol, did not increase Fe(III) reduction, indicating that the reduction rate was not controlled by the energy status of the cell. Control of electron transport at branch points could account for the observed pattern of respiration in the presence of the competing electron acceptors Fe(III) and O₂.     

Light-induced oxidation of the acceptor-side Fe(II) of Photosystem II by exogenous quinones acting through the QB binding site. II. Blockage by inhibitors and their effects on the Fe(III) EPR spectra

Four representative inhibitors of Photosystem II (PS II) Q⁻_A to Q_B electron transfer were shown to bind, at high concentrations, to PS II reaction centers having the acceptor-side non-heme iron in the Fe(III) state. Three of the inhibitors studied, DCMU, o-phenanthroline and dinoseb, modified the EPR spectrum of the Fe(III) relative to that obtained by ferricyanide oxidation in the absence of inhibitor. o-Phenanthroline gave particularly axial symmetry, while DCMU and dinoseb gave more rhombic configurations. The herbicide inhibitor, atrazine and its analogue, terbutryn, had no effect. The dissociation constants for inhibitor binding to reaction centers in the Fe(III) state were measured directly and also estimated from shifts in the midpoint potential of the Fe(III)/Fe(II) couple and were shown to increase by factors of approx. 100, approx. 10 and 10–15 for DCMU (pH 7.5), atrazine (pH 7.0) and o-phenanthroline (pH 7.0), respectively, upon oxidation of the iron. Atrazine and o-phenanthroline, which induce the smallest changes in the midpoint potential of the Fe(III)/Fe(II) couple, were shown to inhibit light-induced oxidation of the Fe(II) by phenyl-p-BQ, described in the preceding paper (Petrouleas, V. and Diner, B.A. (1987) Biochim. Biophys. Acta 893, 126–137). The extent of inhibition was much greater than would be predicted from a simple shift in the midpoint potential for Fe(III)/Fe(II) and we conclude that phenyl-p-BQ and the other quinones, which show light-induced oxidation, act through the Q_B binding site. It is also argued that reduction and oxidation of the iron by ferro- and ferricyanide, respectively, occur through this site. The effects of these inhibitors and of various quinones on the Fe(III) environment are discussed with reference to the known contact points between the protein and o-phenanthroline and terbutryn in the Q_B binding pocket of Rhodopseudomonas viridis reaction centers (Michel, H., Epp, O. and Deisenhofer, J. (1986) EMBO J. 5, 2445–2451). The Fe(III) EPR spectrum is thus a new and sensitive probe of the contact points at which molecules bind to the Q_B binding site.