Functional analysis of the multi-copper oxidase from Legionella pneumophila

Multicopper oxidases have been described to have functions in copper tolerance, manganese oxidation, and iron oxidation in a range of bacteria. The putative cytoplasmic membrane multicopper oxidase from Legionella pneumophila was investigated. The mcoL gene was found to be critical for aerobic extracellular growth under either iron-limiting conditions or in the presence of ferrous Fe(II) iron, as a sole source of this essential metal. The mcoL mutants showed minor growth defects when grown in the presence of Fe(III) as the iron source. In contrast, intracellular growth and survival was not affected by the absence of the mcoL gene regardless of available iron concentration. The evidence presented here could indicate a possible role for mcoL in prevention of the toxic effects of ferrous iron during aerobic conditions. However, a function in high-affinity acquisition of iron could also be possible given the inability of the McoL mutants to grow aerobically under iron-limiting conditions.     

New insights into the protective effect of manganese against oxidative stress

Manganese has emerged as an important trace element in bacterial physiology. The correlation between manganese accumulation and resistance to oxidative stress has led to the suggestion that, in addition to a role as a prosthetic group in superoxide dismutase, manganese could exert its antioxidant effect via non-enzymatic redox reactions. The article by Anjem et al. in the current issue of Molecular Microbiology investigates the role of manganese ions in the defence against hydrogen peroxide in Escherichia coli . The results indicate that the redox activity of manganese is not linked to its protective effect. Instead, it is suggested that manganese replaces ferrous iron in enzymes that contain divalent cations at their active site. This enables the cell to avoid oxidative stress associated with iron release in the presence of hydrogen peroxide.     

Phototrophs in High-Iron-Concentration Microbial Mats: Physiological Ecology of Phototrophs in an Iron-Depositing Hot Spring

At Chocolate Pots Hot Springs in Yellowstone National Park the source waters have a pH near neutral, contain high concentrations of reduced iron, and lack sulfide. An iron formation that is associated with cyanobacterial mats is actively deposited. The uptake of [¹⁴C]bicarbonate was used to assess the impact of ferrous iron on photosynthesis in this environment. Photoautotrophy in some of the mats was stimulated by ferrous iron (1.0 mM). Microelectrodes were used to determine the impact of photosynthetic activity on the oxygen content and the pH in the mat and sediment microenvironments. Photosynthesis increased the oxygen concentration to 200% of air saturation levels in the top millimeter of the mats. The oxygen concentration decreased with depth and in the dark. Light-dependent increases in pH were observed. The penetration of light in the mats and in the sediments was determined. Visible radiation was rapidly attenuated in the top 2 mm of the iron-rich mats. Near-infrared radiation penetrated deeper. Iron was totally oxidized in the top few millimeters, but reduced iron was detected at greater depths. By increasing the pH and the oxygen concentration in the surface sediments, the cyanobacteria could potentially increase the rate of iron oxidation in situ. This high-iron-content hot spring provides a suitable model for studying the interactions of microbial photosynthesis and iron deposition and the role of photosynthesis in microbial iron cycling. This model may help clarify the potential role of photosynthesis in the deposition of Precambrian banded iron formations.     

Oxidation and Reduction of Iron by Acidophilic Bacteria

Abstract

Redox reactions of iron in acidic environments are of economic and environmental significance, for example, for the leaching of metal ores and for the formation of acid mine drainage and acid sulfate soils. Until recently, research on microbial iron metabolism in acidic environments has mainly been focused on the role of aerobic, autotrophic ferrous iron‐oxidizing bacteria. In the present paper, recent new developments in the field of acidophilic iron metabolism are reviewed. In addition to the well‐known autotrophic ferrous iron‐oxidizing organisms, new heterotrophic isolates have been described that are capable of oxidizing ferrous iron. Microorganisms can also play an important role in the reductive part of the iron cycle. Both heterotrophic and autotrophic organisms may also be involved in this process. The contribution of heterotrophic organisms to acidophilic iron cycling can be twofold: In addition to their direct role as a catalyst, these organisms may scavenge organic compounds that inhibit their autotrophic counterparts. Detailed studies of acidophilic ecosystems are needed to assess the significance of the various types of microorganisms for the overall rate of iron cycling in these extreme environments.     

Oxygen toxicity in Streptococcus mutans: manganese, iron, and superoxide dismutase.

When cultured anaerobically in a chemically defined medium that was treated with Chelex-100 to lower its trace metal content, Streptococcus mutans OMZ176 had no apparent requirement for manganese or iron. Manganese or iron was necessary for aerobic cultivation in deep static cultures. During continuous aerobic cultivation in a stirred chemostat, iron did not support the growth rate achieved with manganese. Since the dissolved oxygen level in the chemostat cultures was higher than the final level in the static cultures, manganese may be required for growth at elevated oxygen levels. In medium supplemented with manganese, cells grown anaerobically contained a low level of superoxide dismutase (SOD) activity; aerobic cultivation increased SOD activity at least threefold. In iron-supplemented medium, cells grown anaerobically also had low SOD activity; aerobic incubation resulted in little increase in SOD activity. Polyacrylamide gel electrophoresis of the cell extracts revealed a major band and a minor band of SOD activity in the cells grown with manganese; however, cells grown with iron contained a single band of SOD activity with an Rf value similar to that of the major band found in cells grown with manganese. None of the SOD activity bands were abolished by the inclusion of 2 mM hydrogen peroxide in the SOD activity strain. S. mutans may not produce a separate iron-containing SOD but may insert either iron or manganese into an apo-SOD protein. Alternatively, iron may function in another activity (not SOD) that augments the defense against oxygen toxicity at low SOD levels.     

Phototrophs in high-iron-concentration microbial mats: physiological ecology of phototrophs in an iron-depositing hot spring

At Chocolate Pots Hot Springs in Yellowstone National Park the source waters have a pH near neutral, contain high concentrations of reduced iron, and lack sulfide. An iron formation that is associated with cyanobacterial mats is actively deposited. The uptake of [(14)C]bicarbonate was used to assess the impact of ferrous iron on photosynthesis in this environment. Photoautotrophy in some of the mats was stimulated by ferrous iron (1.0 mM). Microelectrodes were used to determine the impact of photosynthetic activity on the oxygen content and the pH in the mat and sediment microenvironments. Photosynthesis increased the oxygen concentration to 200% of air saturation levels in the top millimeter of the mats. The oxygen concentration decreased with depth and in the dark. Light-dependent increases in pH were observed. The penetration of light in the mats and in the sediments was determined. Visible radiation was rapidly attenuated in the top 2 mm of the iron-rich mats. Near-infrared radiation penetrated deeper. Iron was totally oxidized in the top few millimeters, but reduced iron was detected at greater depths. By increasing the pH and the oxygen concentration in the surface sediments, the cyanobacteria could potentially increase the rate of iron oxidation in situ. This high-iron-content hot spring provides a suitable model for studying the interactions of microbial photosynthesis and iron deposition and the role of photosynthesis in microbial iron cycling. This model may help clarify the potential role of photosynthesis in the deposition of Precambrian banded iron formations.     

Iron uptake by the microaerophilic anaerobe Bifidobacterium bifidum var. pennsylvanicus

A system was designed to investigate ferrous iron transport into Bifidobacterium bifidum var. pennsylvanicus. It involved the incubation of the organisms with labeled ferrous iron in the Norris medium at pH 5, in which the bacteria had grown. Iron uptakes were similar under aerobic and anaerobic conditions. Ferrous but not ferric iron was taken up by the organisms. Iron uptake showed saturation kinetics and a marked temperature dependence. 2,4-Dinitrophenol and thenoltrifluoroacetate but not azide or trypsin treatment inhibited iron uptake. Zinc inhibited iron uptake competitively. Iron uptake from used medium was much greater than that from fresh medium at the same pH. It is concluded that ferrous iron uptake by the microorganisms is a carrier-mediated active phenomenon, inhibited by zinc, which may involve a substance elaborated into the medium by the organism.     

Insects, oxygen, and iron

Konrad Bloch developed an interest in insects because they are unable to make sterols, and in yeast because these cells need oxygen to make sterols and unsaturated fatty acids. Insects, like all other organisms, must deal with the toxic effects of oxygen in the presence of iron, which itself is a vital nutrient. They do so by making proteins with high affinity for ferric or ferrous ions. Two such proteins are transferrins and ferritins. Insects produce both of these proteins, but use them in different ways from most other organisms. Insect transferrins appear to be involved in innate immunity, perhaps by sequestering ferric ions to prevent pathogens and parasites from utilizing them. Insect ferritins, unlike those of any other group of organisms, are exported into the extracellular space (hemolymph). They may be involved in iron transport and/or protection against iron overload in the diet.     

Genetic and biochemical analysis of high iron toxicity in yeast: iron toxicity is due to the accumulation of cytosolic iron and occurs under both aerobic and anaerobic conditions

Iron storage in yeast requires the activity of the vacuolar iron transporter Ccc1. Yeast with an intact CCC1 are resistant to iron toxicity, but deletion of CCC1 renders yeast susceptible to iron toxicity. We used genetic and biochemical analysis to identify suppressors of high iron toxicity in Δccc1 cells to probe the mechanism of high iron toxicity. All genes identified as suppressors of high iron toxicity in aerobically grown Δccc1 cells encode organelle iron transporters including mitochondrial iron transporters MRS3, MRS4, and RIM2. Overexpression of MRS3 suppressed high iron toxicity by decreasing cytosolic iron through mitochondrial iron accumulation. Under anaerobic conditions, Δccc1 cells were still sensitive to high iron toxicity, but overexpression of MRS3 did not suppress iron toxicity and did not result in mitochondrial iron accumulation. We conclude that Mrs3/Mrs4 can sequester iron within mitochondria under aerobic conditions but not anaerobic conditions. We show that iron toxicity in Δccc1 cells occurred under both aerobic and anaerobic conditions. Microarray analysis showed no evidence of oxidative damage under anaerobic conditions, suggesting that iron toxicity may not be solely due to oxidative damage. Deletion of TSA1, which encodes a peroxiredoxin, exacerbated iron toxicity in Δccc1 cells under both aerobic and anaerobic conditions, suggesting a unique role for Tsa1 in iron toxicity.     

Hydrogen peroxide and the evolution of oxygenic photosynthesis

The early atmosphere of the Earth is considered to have been reducing (H₂ rich) or neutral (CO₂-N₂). The present atmosphere by contrast is highly oxidizing (20% O₂). The source of this oxygen is generally agreed to have been oxygenic photosynthesis, whereby organisms use water as the electron donor in the production of organic matter, liberating oxygen into the atmosphere. A major question in the evolution of life is how oxygenic photosynthesis could have evolved under anoxic conditions — and also when this capability evolved. It seems unlikely that water would be employed as the electron donor in anoxic environments that were rich in reducing agents such as ferrous or sulfide ions which could play that role. The abiotic production of atmospheric oxidants could have provided a mechanism by which locally oxidizing conditions were sustained within spatially confined habitats thus removing the available reductants and forcing photosynthetic organisms to utilize water as the electron donor. We suggest that atmospheric H₂O₂ played the key role in inducing oxygenic photosynthesis because as peroxide increased in a local environment, organisms would not only be faced with a loss of reductant, but they would also be pressed to develop the biochemical apparatus (e.g., catalase) that would ultimately be needed to protect against the products of oxygenic photosynthesis. This scenario allows for the early evolution of oxygenic photosynthesis while global conditions were still anaerobic.     

Old Iron, Young Copper: from Mars to Venus

Iron and copper are metals which play an important role in the living world. From a brief consideration of their chemistry and biochemistry we conclude that the early chemistry of life used water soluble ferrous iron while copper was in the water-insoluble Cu(I) state as highly insoluble sulphides. The advent of oxygen was a catastrophic event for most living organisms, and can be considered to be the first general irreversible pollution of the earth. In contrast to the oxidation of iron and its loss of bioavailability as insoluble Fe(III), the oxidation of insoluble Cu(I) led to soluble Cu(II). A new iron biochemistry became possible after the advent of oxygen, with the development of chelators of Fe(III), which rendered iron once again accessible, and with the control of the potential toxicity of iron by its storage in a water soluble, non-toxic, bio-available storage protein (ferritin). Biology also discovered that whereas enzymes involved in anaerobic metabolism were designed to operate in the lower portion of the redox spectrum, the arrival of dioxygen created the need for a new redox active metal which could attain higher redox potentials. Copper, now bioavailable, was ideally suited to exploit the oxidizing power of dioxygen. The arrival of copper also coincided with the development of multicellular organisms which had extracellular cross-linked matrices capable of resisting attack by oxygen free radicals. After the initial `iron age' subsequent evolution moved, not towards a `copper age', but rather to an `iron-copper' age. In the second part of the review, this symbiosis of iron and copper is examined in yeast. We then briefly consider iron and copper metabolism in mammals, before looking at iron-copper interactions in mammals, particularly man, and conclude with the reflection that, as in Greek and Roman mythology, a better understanding of the potentially positive interactions between Mars (iron) and Venus (copper) can only be to the advantage of our species.     

A Streptococcus mutans superoxide dismutase that is active with either manganese or iron as a cofactor

The superoxide dismutase produced by Streptococcus mutans OMZ176 during aerobic growth in a chemically defined medium (modified FMC) that was treated with Chelex 100 (to lower trace metal contamination) and supplemented with high purity manganese was purified (162-fold) by heat treatment, ammonium sulfate precipitation, and chromatofocusing chromatography. The superoxide dismutase produced during aerobic growth in the same medium, but without manganese and supplemented with high purity iron, was similarly purified (220-fold). The molecular masses of each holoenzyme were approximately 43,000 with a subunit mass of 20,700, indicating that the enzymes were dimers of two equally sized subunits. The superoxide dismutase from manganese-grown cells was a manganese enzyme (MnSOD) containing 1.2 atoms of manganese and 0.25 atoms of iron/subunit. The superoxide dismutase from iron-grown cells was an iron enzyme (FeSOD) containing 0.07 atoms of manganese and 0.78 atoms of iron/subunit. The amino acid compositions of the MnSOD and the FeSOD were virtually identical, and their amino-terminal sequences were identical through the first 22 amino acids. Dialysis of the FeSOD with o-phenanthroline and sodium ascorbate generated aposuperoxide dismutase with 94% loss of activity; subsequent dialysis of apoenzyme with either manganese sulfate or ferrous sulfate reconstituted activity (recoveries of 37 and 30%, respectively). Electrophoretic determination of cytoplasmic radioiron distribution indicated that (during aerobic growth) manganese prevented insertion of iron into superoxide dismutase, although the iron levels of at least two other cytoplasmic fractions were not altered by manganese. Therefore, S. mutans used the same aposuperoxide dismutase to form either FeSOD or MnSOD, depending upon which metal was available in the culture medium. Such "cambialistic" enzymes (those capable of making a cofactor substitution) may represent a previously unrecognized family of superoxide dismutases.     

On the effect of ferrous sulphate on the available manganese in the soil and the uptake of manganese by the plant II

Summary Pot experiments with oats on manganese deficient sandy and moor soils, which are not deficient in iron, showed a steady increase in yield and manganese uptake by the plants with increasing additions of ferrous sulphate at four levels of manganese. Residual effects from ferrous sulphate application were not found either in the case of yields or manganese uptake by oats on a sandy soil.The effect observed following application of ferrous sulphate was due to a manganese effect. This is corroborated by the coincidence of the yield curves, showing the relationship between absorbed manganese and yield of dry matter, following the application of manganese sulphate and ferrous sulphate.Soils treated withM magnesium nitrate in the presence of equivalent quantities of either ferrous sulphate or hydroquinone yielded the same amounts of manganese. This result suggests that the manganese effect of ferrous sulphate is due to reduction of higher manganese oxides by ferrous sulphate.     

Iron in evolution

Iron chemistry in the environment and in organisms is entwined. The iron surface minerals in solution for the first billion years of the planet were ferrous compounds. This ion became and has remained a major participant in organisms. The evolution of iron was due to its oxidation to insoluble ferric ions by oxygen released from organisms. The evolution of cellular iron chemistry then required uptake from this oxidised state. Use was expanded from the mainly electron transfer properties in the original reductive cell interior to employment in external oxidative chemistry. The environment/organisms evolution is that of one predictable chemical system.     

光合作用氧释放机理研究进展

植物在光合作用过程中不仅为同化CO2提供能量和还原力,同时裂解水放出氧气。放氧反应主要由光系统Ⅱ(PSⅡ)氧化侧的4个锰原子组成的锰簇催化完成的。因此,锰簇在光合放氧过程中起看至关重要的作用。文章概述了对锰簇及其微环境的结构和功能的研究进展。     

Mechanism of photosynthetic water oxidation in the dimeric oxygen-evolving complex of chloroplast photosystem II

Based on the analysis of the molecular organization and properties of an isolated oxygen-evolving complex of photosystem II of plant chloroplasts, a mechanism of water oxidation and oxygen release during photosynthesis was proposed. It is suggested that the photolysis of water occurs in a dimeric oxygen-evolving complex consisting of two core complexes. In the region of contact of these complexes, a hydrophobic "boiler" is formed where the conditions for screening and stabilization of Z-linanded manganese cations accumulating positive charges for the oxidation of water molecules are created. A prerequisite to the photolysis of water is the formation of a binuclear [Mn(3+)-OH ... HO-Mn3+] hydroxyl-manganese associate, which appears in the dimeric oxygen-evolving complex after the first two light flashes as a result of photohydrolysis of photochemically oxidized Z-liganded manganese cations. The process is accompanied by the release of the first water protons to the medium. The photosynthetic oxidation of water hydroxyls occurs at the next stage and is considered as synchronous detachment of four electrons from two bound OH-groups of the associate upon photooxidation of Mn3+ cations to Mn4+ cations after two subsequent light flashes. This process is accompanied by the disproportionation of electron density and the formation of a bond between oxygen atoms of hydroxyls followed by the evolution of molecular oxygen and protons, and regeneration of two starting Mn2+ cations and the primary state of the system.     

Studies on the growth ofThiobacillus ferrooxidans

Summary Uranyl sulphate (0.2–0.9 mM) inhibited ferrous iron oxidation by growing cultures ofThiobacillus ferrooxidans. The addition of 5–100 mM uranium to the cultures caused immediate cessation of carbon dioxide fixation, rapid loss of viability and gradual depression of ferrous iron oxidation. Virtually no uranium was found in washed cells grown in the presence of subtoxic to toxic amounts of uranyl sulphate. Uranium-poisoned organisms appeared plasmolyzed in electron micrographs. Cultures tolerant to 5 mM UO₂ ²⁺ were develoepd by successive subculturing in increased uranium concentrations. The tolerance was maintained during subculturing in uranium-free medium. Frequency of mutants resistant to 1.0 and 1.5 mM UO₂ ²⁺ was 1 per 1.3×10⁶ and 1 per 9.0×10⁸, respectively. The frequency was increased in the presence of 15–150 mM nickel, zinc and manganese. In liquid cultures, bivalent cations and EDTA alleviated the toxicity of 2 mM uranyl sulphate.     

Lethal oxidative damage and mutagenesis are generated by iron in delta fur mutants of Escherichia coli: protective role of superoxide dismutase.

The Escherichia coli Fur protein, with its iron(II) cofactor, represses iron assimilation and manganese superoxide dismutase (MnSOD) genes, thus coupling iron metabolism to protection against oxygen toxicity. Iron assimilation is triggered by iron starvation in wild-type cells and is constitutive in fur mutants. We show that iron metabolism deregulation in fur mutants produces an iron overload, leading to oxidative stress and DNA damage including lethal and mutagenic lesions. fur recA mutants were not viable under aerobic conditions and died after a shift from anaerobiosis to aerobiosis. Reduction of the intracellular iron concentration by an iron chelator (ferrozine), by inhibition of ferric iron transport (tonB mutants), or by overexpression of the iron storage ferritin H-like (FTN) protein eliminated oxygen sensitivity. Hydroxyl radical scavengers dimethyl sulfoxide and thiourea also provided protection. Functional recombinational repair was necessary for protection, but SOS induction was not involved. Oxygen-dependent spontaneous mutagenesis was significantly increased in fur mutants. Similarly, SOD deficiency rendered sodA sodB recA mutants nonviable under aerobic conditions. Lethality was suppressed by tonB mutations but not by iron chelation or overexpression of FTN. Thus, superoxide-mediated iron reduction was responsible for oxygen sensitivity. Furthermore, overexpression of SOD partially protected fur recA mutants. We propose that a transient iron overload, which could potentially generate oxidative stress, occurs in wild-type cells on return to normal growth conditions following iron starvation, with the coupling between iron and MnSOD regulation helping the cells cope.     

Oxidative stress and neurodegeneration: the involvement of iron

Many evidences indicate that oxidative stress plays a significant role in a variety of human disease states, including neurodegenerative diseases. Iron is an essential metal for almost all living organisms due to its involvement in a large number of iron-containing proteins and enzymes, though it could be also toxic. Actually, free iron excess generates oxidative stress, particularly in brain, where anti-oxidative defences are relatively low. Its accumulation in specific regions is associated with pathogenesis in a variety of neurodegenerative diseases (i.e., Parkinson’s disease, Alzheimer’s disease, Huntington’s chorea, Amyotrophic Lateral Sclerosis and Neurodegeneration with Brain Iron Accumulation). Anyway, the extent of toxicity is dictated, in part, by the localization of the iron complex within the cell (cytosolic, lysosomal and mitochondrial), its biochemical form, i.e., ferritin or hemosiderin, as well as the ability of the cell to prevent the generation and propagation of free radical by the wide range of antioxidants and cytoprotective enzymes in the cell. Particularly, ferrous iron can act as a catalyst in the Fenton reaction that potentiates oxygen toxicity by generating a wide range of free radical species, including hydroxyl radicals (·OH). The observation that patients with neurodegenerative diseases show a dramatic increase in their brain iron content, correlated with the production of reactive oxigen species in these areas of the brain, conceivably suggests that disturbances in brain iron homeostasis may contribute to the pathogenesis of these disorders. The aim of this review is to describe the chemical features of iron in human beings and iron induced toxicity in neurodegenerative diseases. Furthermore, the attention is focused on metal chelating drugs therapeutic strategies.