Inhibition of enzymatic cellulolysis by phenolic compounds

Phenolics derived from lignin and other plant components can pose significant inhibition on enzymatic conversion of cellulosic biomass materials to useful chemicals. Understanding the mechanism of such inhibition is of importance for the development of viable biomass conversion technologies. In native plant cell wall, most of the phenolics and derivatives are found in polymeric lignin. When biomass feedstocks are pretreated (prior to enzymatic hydrolysis), simple or oligomeric phenolics and derivatives are often generated from lignin modification/degradation, which can inhibit biomass-converting enzymes. To further understand how such phenolic substances may affect cellulase reaction, we carried out a comparative study on a series of simple and oligomeric phenolics representing or mimicking the composition of lignin or its degradation products. Consistent to previous studies, we observed that oligomeric phenolics could exert more inhibition on enzymatic cellulolysis than simple phenolics. Oligomeric phenolics could inactivate cellulases by reversibly complexing them. Simple and oligomeric phenolics could also inhibit enzymatic cellulolysis by adsorbing onto cellulose. Individual cellulases showed different susceptibility toward these inhibitions. Polyethylene glycol and tannase could respectively bind and degrade the studied oligomeric phenolics, and by doing so mitigate the oligomeric phenolic's inhibition on cellulolysis.     

Age-related accumulation of non-heme ferric and ferrous iron in mouse ovarian stroma visualized by sensitive non-heme iron histochemistry

Sensitive non-heme iron histochemistry--namely, the perfusion-Perls method and perfusion-Turnbull method--was applied to study the distribution and age-related accumulation of non-heme ferric iron and ferrous iron in mouse ovary. Light and electron microscopic studies revealed that non-heme ferric iron is distributed predominantly in stromal tissue, especially in macrophages. By contrast, the distribution of non-heme ferrous iron was restricted to a few ovoid macrophages. Aged ovaries exhibited remarkable non-heme iron accumulation in all stromal cells. In particular, non-heme ferrous iron level was increased in stromal tissue, suggestive of increased levels of redox-active iron, which can promote oxidative stress. Moreover, intense localization of both non-heme ferric and ferrous iron was observed in aggregated large stromal cells that were then characterized as ceroid-laden enlarged macrophages with frothy cytoplasm. Intraperitoneal iron overload in adult mice resulted in non-heme iron deposition in the entire stroma and generation of enlarged macrophages, suggesting that excessive iron accumulation induced macrophage morphological changes. The data indicated that non-heme iron accumulation in ovarian stromal tissue may be related to aging of the ovary due to increasing oxidative stress.     

Separate pathways for cellular uptake of ferric and ferrous iron

Separate pathways for transport of nontransferrin ferric and ferrous iron into tissue cultured cells were demonstrated. Neither the ferric nor ferrous pathway was shared with either zinc or copper. Manganese shared the ferrous pathway but had no effect on cellular uptake of ferric iron. We postulate that ferric iron was transported into cells via beta(3)-integrin and mobilferrin (IMP), whereas ferrous iron uptake was facilitated by divalent metal transporter-1 (DMT-1; Nramp-2). These conclusions were documented by competitive inhibition studies, utilization of a beta(3)-integrin antibody that blocked uptake of ferric but not ferrous iron, development of an anti-DMT-1 antibody that blocked ferrous iron and manganese uptake but not ferric iron, transfection of DMT-1 DNA into tissue culture cells that showed enhanced uptake of ferrous iron and manganese but neither ferric iron nor zinc, hepatic metal concentrations in mk mice showing decreased iron and manganese but not zinc or copper, and data showing that the addition of reducing agents to tissue culture media altered iron binding to proteins of the IMP and DMT-1 pathways. Although these experiments show ferric and ferrous iron can enter cells via different pathways, they do not indicate which pathway is dominant in humans.     

The role of ferrichrome reductase in iron metabolism of Ustilago sphaerogena.

Ferrichrome, the ferric ionophore for Ustilago sphaerogena, can serve as a source of iron for the enzyme ferrochelatase (protoheme ferrolyase, EC 4.99.1.1) in this organism, but only after enzymatic removal of the iron from its carrier. U. sphaerogena contains a specific ferrichrome reductase (NADH:ferrichrome oxidoreductase) which catalyzes cellular dissociation of the complex by reduction of the metal to the ferrous state. A spectrophotometric assay was developed based on trapping of the ferrous ion produced by ferrozine. There is an apparent inhibition by oxygen which is thought to be due to re-oxidation of the metal under the assay conditions. The close structural analogue, ferrichrome A, is not a substrate, nor is the ester type siderochrome ferric hexahydro-N,N',N"-triacetylfusarinine C. Aluminum desferriferrichrome is inhibitory. The importance of this enzyme for the metabolism of iron in this organism is discussed.     

Influence of histidine on lipid peroxidation in sarcoplasmic reticulum.

The free amino acid, histidine, which exists at high concentrations in some muscle systems, has previously been demonstrated to both inhibit and activate lipid peroxidation in membrane model systems. This study sought to characterize the specificity of histidine's effect on iron-catalyzed enzymatic and nonenzymatic lipid peroxidation. Under conditions of activation (histidine added to the reaction mixture after ADP and ferric ion), alpha-amino, carboxylate, and pyrrole nitrogen were demonstrated to be involved by kinetic techniques in the activation of the enzymatic system. It is hypothesized that a mixed ligand complex (iron, ADP, and histidine) formed may allow rapid redox cycling of iron. While increasing concentrations of histidine led to increasing levels of stimulation in the enzymatic system, the maximum stimulation of a nonenzymatic lipid peroxidation system of ascorbate and ferric ion occurred at histidine concentrations near 2.5 mM. Inhibition of a nonenzymatic system (ferrous ion), on the other hand, occurred at all concentrations of histidine when the ferrous ion was exposed to ADP prior to histidine. In enzymatic systems, under conditions when the ferric ion was exposed to histidine prior to ADP, inhibition of lipid peroxidation by histidine also occurred. The inhibitory effect of histidine was ascribed to the imidazole group and may arise from the formation of a different iron complex or the acceleration of polymerization, dehydration, and insolubilization of the ferric ion by the imidazole nitrogen. The demonstrated ability of histidine to affect in vitro lipid peroxidation systems raises the possibility that this free amino acid may modulate lipid peroxidation in vivo.     

Iron metabolism in anoxic environments at near neutral pH

Anaerobic dissimilatory ferric iron-reducing and ferrous iron-oxidizing bacteria gain energy through reduction or oxidation of iron minerals and presumably play an important role in catalyzing iron transformations in anoxic environments. Numerous ferric iron-reducing bacteria have been isolated from a great diversity of anoxic environments, including sediments, soils, deep terrestrial subsurfaces, and hot springs. In contrast, only few ferrous iron-oxidizing bacteria are known so far. At neutral pH, iron minerals are barely soluble, and the mechanisms of electron transfer to or from iron minerals are still only poorly understood. In natural habitats, humic substances may act as electron carriers for ferric iron-reducing bacteria. Also fermenting bacteria were shown to channel electrons to ferric iron via humic acids. Whether quinones or cytochromes released from cells act as electron transfer components in ferric iron reduction is still a matter of debate. Anaerobic ferrous iron-oxidizing phototrophic bacteria, on the other hand, appear to excrete complexing agents to prevent precipitation of ferric iron oxides at their cell surfaces. The present review evaluates recent findings on the physiology of ferric iron-reducing and ferrous iron-oxidizing bacteria with respect to their relevance to microbial iron transformations in nature.     

Evaluation of the metal ion requirement of the human deoxyhypusine hydroxylase from HeLa cells using a novel enzyme assay

Hypusine synthesis in the eukaryotic initiation factor 5A is a unique two-step posttranslational modification. After deoxyhypusine is generated by the deoxyhypusine synthase, the deoxyhypusine hydroxylase (EC 1.14.99.29) catalyzes the formation of mature hypusine. A rapid assay for monitoring the deoxyhypusine hydroxylase activity was established, employing the oxidative cleavage of the hypusyl residue and subsequent extraction of the generated aldehydes. As metal ion chelators have been reported to inhibit the deoxyhypusine hydroxylase, the mechanism of this inhibition and the effect of transition metal ions on the enzyme activity were investigated. A ferric ion appears to be essential for enzymatic activity, the inhibition of which is entirely attributed to the metal ion bunding capacity of the chelators.     

Macrophage erythrophagocytosis and iron exocytosis

SUMMARY

Senescent, or damaged, erythrocytes are removed from the blood stream mainly by the macrophage system. Such cells may acquire and store large quantities of the redox-active transition metal iron that, if released together with superoxide and hydrogen peroxide during an oxidative burst, may induce peroxidative reactions with a variety of surrounding substances, e.g., low-density lipoprotein (LDL). In this study we demonstrate 1. the temporary sequestration of iron within the secondary lysosomal apparatus of both established macrophage-like J-774 cells and human monocyte-derived macrophages secondary to the uptake and degradation of native and photo-oxidized (ultraviolet UV light) erythrocytes; and 2. an ensuing development by these cells of a capacity for iron-exocytosis. The binding and uptake by human macrophages and J-774 cells of artificially aged, UV-irradiated erythrocytes were stimulated compared to that of native erythrocytes. The uptake resulted in lysosomal accumulation of iron in a low-molecular weight form, as shown by autometallography. Cells exposed to ferric chloride were used as positive controls. Ensuing exocytosis of iron to the culture medium was demonstrated by atomic absorption spectroscopy. Our findings suggest that macrophage erythrophagocytosis is a useful model for the study of the sequestration of iron within the macrophage acidic vacuolar apparatus, its subsequent exocytosis, and oxidative effect on extracellular LDL.     

Reduction of ferric iron by acidophilic heterotrophic bacteria: evidence for constitutive and inducible enzyme systems in Acidiphilium spp

AIMS: To compare the abilities of two obligately acidophilic heterotrophic bacteria, Acidiphilium acidophilum and Acidiphilium SJH, to reduce ferric iron to ferrous when grown under different culture conditions. METHODS AND RESULTS: Bacteria were grown in batch culture, under different aeration status, and in the presence of either ferrous or ferric iron. The specific rates of ferric iron reduction by fermenter-grown Acidiphilium SJH were unaffected by dissolved oxygen (DO) concentrations, while iron reduction by A. acidophilum was highly dependent on DO concentrations in the growth media. The ionic form of iron present (ferrous or ferric) had a minimal effect on the abilities of harvested cells to reduce ferric iron. Whole cell protein profiles of Acidiphilium SJH were very similar, regardless of the DO status of the growth medium, while additional proteins were present in A. acidophilum grown microaerobically compared with aerobically-grown cells. CONCLUSIONS: The dissimilatory reduction of ferric iron is constitutive in Acidiphilium SJH while it is inducible in A. acidophilum. SIGNIFICANCE AND IMPACT OF THE STUDY: Ferric iron reduction by Acidiphilium spp. may occur in oxygen-containing as well as anoxic acidic environments. This will detract from the effectiveness of bioremediation systems where removal of iron from polluted waters is mediated via oxidation and precipitation of the metal.     

The effect of ferric ion on the rate of ferrous oxidation by Thiobacillus ferrooxidans

 In this study, the effect of ferric ion and cell concentrations on the oxidation of ferrous ion by T. ferrooxidans was investigated. Ferric ions competitively inhibited ferrous ion oxidation by the bacteria. The inhibitory effect of ferric ion was, however, reduced by increasing cell concentration. The apparent ferric ion inhibition constant did not change with increasing cell concentration. The ferrous ion oxidation kinetics in the absence and presence of ferric ion changes from the standard Michaelis-Menten type at low cell concentrations to pseudo-first-order kinetics at high cell concentration. Received: 8 August 1995/Received revision: 31 October 1995/Accepted: 10 November 1995     

Cellular regulation of iron assimilation

Cells of plants, most microorganisms, and animals require well-defined amounts of iron for survival, replication, and differentiation. The metal is an important component of such processes as synthesis of DNA, RNA, and chlorophyll; electron transport; oxygen metabolism; and nitrogen fixation. Because of the insolubility of iron in aerobic environments at neutral and alkaline pH values, cells have had to devise specific strategies to assimilate the metal. These include (1) development of systems for reducing ferric ions to the more soluble ferrous ions at the cell surface, (2) employment of small carrier molecules (termed siderophores) that have high affinity for ferric ions and receptor proteins for the ferrated molecules, and (3) use of transferrin and other proteins that can transport ferric ions. Excessive amounts of iron are toxic, however, and intracellular storage capacity is limited and efflux mechanisms generally are lacking. Thus, cells have had to develop methods of preventing over-accumulation of the metal. These include use of (1) oxygen to convert ferrous to ferric ions, (2) small molecules that can bind ferrous ions, termed siderophraxes, and (3) proteins that, when combined with ferrous ions, repress the expression of iron transport genes. Often, one organism can prevent growth of neighbors by restricting their access to iron. In other cases, cells assist each other by sharing iron acquisition systems or by restricting influx of excess iron. Homeostatic control of other essential trace metals also is required for optimal cell function. Nevertheless, since iron thus far has received most attention, it serves as the model of mineral metabolism. Moreover, many of the observations made on control of iron metabolism suggest possible applications in prevention and management of plant and animal infections as well as of neoplastic diseases, arthropathy, and cardiomyopathy. This review will focus on (1) problems at the cellular level of iron acquisition, storage, and exclusion; and (2) the strategies devised by cells of plants, microorganisms, and animals to solve these problems.     

Role of iron in particulate methane monooxygenase from Methylosinus trichosporium OB3b

The effect of iron ions on particulate methane monooxygenase was studied by using the EDTA-treated membranes from Methylosinus trichosporium OB3b. When the membrane was treated with EDTA the activity remained 82% of the as-isolated membranes, and the activity of the EDTA-treated membranes was strongly influenced by the addition of metal ions. Among the metal ions, ferric, ferrous and cupric ions stimulated the activity, indicating those ions were needed for the activity. When propargylamine was added, pMMO activity decreased and also the iron ESR signal decreased. As the ESR signal involves the ferrous nitrosyl complex in EDTA-treated membranes, the active site of pMMO may contain a mononuclear non-heme iron.     

The reaction of ferric- and ferrous salts with bleomycin.

1. A comparative study shows that ferrous ions give a much better yield of Fe(III)-bleomycin than ferric ions, when iron salt is added to bleomycin in a buffer solution (pH 7.2). 2. The amount of Fe(III)-bleomycin formed after addition of ferric ions was markedly increased in the presence of ferric ion binding compounds (BSA, citrate) or reducing agents (ascorbate, cysteine).     

Cytochrome P450: substrate and prosthetic-group free radicals generated during the enzymatic cycle

During the enzymatic cycle of the cytochromes P450, dioxygen binds to the ferrous haemprotein when the resting ferric haemprotein has undergone a one-electron oxidation after substrate binding. A further one-electron reduction generates an intermediate that is isoelectronic with a peroxide dianion coordinated to a ferric iron. Heterolytic cleavage of the omicron--omicron bond generates water and a species which is formally an oxene (oxygen atom) coordinated by iron(III). However, on the basis of model reactions and by analogy to the catalases and peroxidases, this active oxidizing intermediate is formulated as an oxo-FeIV porphyrin pi-cation radical. The radical is stabilized by delocalization on the porphyrin macrocycle and the high oxidation state is achieved by oxidizing both the metal and the porphyrin ring of the haemprotein. Hydrogen atom abstraction from a saturated hydrocarbon substrate generates a substrate free radical, constrained by the protein binding site, and the equivalent of a hydroxyl radical bound to iron(III). Coupling of the 'hydroxy' and substrate radicals generates hydroxylated product and resting protein. For olefins an initial electron transfer to oxidized haemprotein gives a substrate cation radical. Further reaction of this radical can give the epoxide, the principal product; an aldehyde or ketone by rearrangement; or an alkylated haemprotein resulting in suicide inhibition.     

Influence of ionizing radiation, application of iron ions and their chelate complexes on the oxidative status of blood serum of rats

Influence of ionizing radiation, ions of iron and their chelate complexes on the oxidative status of blood serum of rats has been investigated. Animals were irradiated by gamma-rays 60Co at a dose of 4 Gy. Ions of iron and iron chelates with nitrilotriacetic acid and citric acid were introduced into animals intra-abdominally at a doze of 10 mg of iron on 1 kg of body weight. The oxidative status of blood serum was determined according to the estimated content of oxidizing peroxide equivalents which oxidize ferrous iron in ferric iron with the subsequent estimation of ferric iron by means of xylenol orange. We also estimated the total content of iron in blood serum using ferrozine as an indicator. The oxidative status was defined 24 and 96 hours after irradiation and 2 hours after introduction of iron ions and their chelates. The research conducted has shown that the concentration of oxidizing peroxide equivalents in serum and the total iron concentration increase 1.47 times and 1.63 times correspondingly 24 hours after irradiation. The increase in the content of oxidizing peroxide equivalents and iron owing to Fenton's reaction can lead to the appearance of OH* radical and raise the level of damage of nuclear and membrane structures in irradiated cells. 2 hours after introduction of iron ions and their chelates, the content of oxidizing peroxide equivalents increased in the blood serum of irradiated and non-irradiated rats, and the maximum effect was observed when introducing ferrous iron and its chelate with citric acid.     

Modeling continuous bioleach reactors

The results of recent research have shown that the bioleaching of sulfide minerals occurs via a two‐step mechanism. In this mechanism, the sulfide mineral is chemically oxidized by the ferric‐iron in the bioleaching liquor. The ferrous‐iron produced is subsequently oxidized to ferric‐iron by the microorganism. Further research has shown that the rates of both the ferric leaching and ferrous‐iron oxidation are governed by the ferric/ferrous‐iron ratio (i.e., the redox potential). During the steady‐state operation of a bioleach reactor, the rate of iron turnover between the chemical ferric leaching of the mineral and the bacterial oxidation of the ferrous‐iron will define the rate and the redox potential at which the system will operate. The balance between the two rates will in turn depend on the species used, the microbial concentration, the residence time employed, the nature of the sulfide mineral being leached, and its active surface area. The model described proposes that the residence time and microbial species present determine the microbial growth rate, which in turn determines the redox potential in the bioleach liquor. The redox potential of the solution, in turn, determines the degree of leaching of the mineral; that is, conversion in the bioleach reactor. © 1999 John Wiley & Sons, Inc. Biotechnol Bioeng 64: 671–677, 1999.     

固定化Acidithiobacillus ferrooxidans对Fe2+氧化及其生物反应器研究进展

Acidithiobacillus ferrooxidans 对Fe2+的生物氧化是一个非常重要的反应过程, 在生物浸矿、H2S等废气的脱硫、含重金属污泥和酸性矿坑废水的处理等领域有着重要的应用。近些年来,大量的研究主要集中A. ferrooxidans及其反应过程等方面,然而,A. ferrooxidans对Fe2+的催化氧化速率缓慢和稳定性欠佳等问题仍然限制了其商业应用。因此,对A. ferrooxidans的固定化及其生物反应器研究是该技术进一步发展的关键。本文评述了A. ferrooxidans最新应用、存在的问题和解决办法,重点比较了目前文献中报道的各种A. ferrooxidans固定材料、方法,并对目前采用的各种固定化A. ferrooxidans生物反应系统的效率和结构等方面进行了讨论和分析。     

Relief of Casein Inhibition of Bacillus stearothermophilus by Iron, Calcium, and Magnesium

Growth of Bacillus stearothermophilus strain NCA 1518 Smooth in Dextrose Tryptone Agar (DTA) was inhibited by sodium caseinate. Binding studies indicated that sodium caseinate, when present in DTA, had the capacity to effect an iron deficiency which could cause inhibition of growth. Additions of essential cations, iron (1 mM), calcium (5 mM), magnesium (10 mM), or hydrogen ion (pH 5.7), relieved inhibition. Responses to and interactions among these relief factors were analyzed statistically. Equations were fitted to the data and were used to estimate responses to all treatment combinations within the ranges tested. Results from these studies indicated that calcium, magnesium, and hydrogen ion acted by decreasing the binding capacity of the protein for iron, rendering this metal available for metabolic needs. Evidence was obtained that ferrous rather than ferric iron was the limiting factor in DTA containing sodium caseinate.     

Interaction of alpha-tocopherol with iron: antioxidant and prooxidant effects of alpha-tocopherol in the oxidation of lipids in aqueous dispersions in the presence of iron

The dual functions of alpha-tocopherol in the oxidation of lipids in aqueous dispersions in the presence of iron were studied, aiming specifically at elucidating the effect of interaction between alpha-tocopherol and iron. Ferrous ion decomposed hydroperoxide rapidly and induced the free radical chain oxidation of soybean phosphatidylcholine liposomes. alpha-Tocopherol acted primarily as a radical scavenger in the oxidation induced by ferrous ion and acted as an antioxidant. Ferric ion decomposed hydroperoxide much more slowly than ferrous ion, but it also induced the oxidation of liposomal membranes. alpha-Tocopherol incorporated into artificial liposomal membranes reduced ferric ion rapidly to give more reactive ferrous ion, and alpha-tocopherol acted either as an antioxidant or as a prooxidant depending on the experimental conditions. When alpha-tocopherol was depleted by the interaction with ferric ion, it acted solely as a prooxidant, whereas if some alpha-tocopherol remained, it acted as an antioxidant. On the other hand, alpha-tocopherol residing in the intact erythrocyte membranes did not reduce ferric ion in the aqueous region.     

Ferric iron and superoxide ions are required for the killing of cultured hepatocytes by hydrogen peroxide. Evidence for the participation of hydroxyl radicals formed by an iron-catalyzed Haber-Weiss reaction

Cultured hepatocytes pretreated with the ferric iron chelator deferoxamine were resistant to the toxicity of H2O2 generated by either glucose oxidase or by the metabolism of menadione (2-methyl-1,4-naphthoquinone). Ferric, ferrous, or cupric ions restored the sensitivity of the cells to H2O2. Deferoxamine added to hepatocytes previously treated with this chelator prevented the restoration of cell killing by only ferric iron. The free radical scavengers mannitol, thiourea, benzoate, and 4-methylmercapto-2-oxobutyrate protected either native cells exposed to H2O2 or pretreated hepatocytes exposed to H2O2 and given ferric or ferrous iron. Superoxide dismutase prevented the killing of native hepatocytes by either glucose oxidase or menadione. With deferoxamine-pretreated hepatocytes, superoxide dismutase prevented the cell killing dependent upon the addition of ferric but not ferrous iron. Catalase prevented the killing by menadione of deferoxamine-pretreated hepatocytes given either ferric or ferrous iron. Deferoxamine pretreatment did not prevent the toxicity of t-butyl hydroperoxide but did, however, prevent that of cumene hydroperoxide. It is concluded that both ferric iron and superoxide ions are required for the killing of cultured hepatocytes by H2O2. The toxicity of H2O2 is also dependent upon its reaction with ferrous iron to form hydroxyl radicals by the Fenton reaction. The ferrous iron needed for this reaction is formed by the reduction of cellular ferric iron by superoxide ions. Such a sequence corresponds to the so-called iron-catalyzed Haber-Weiss reaction, and the present report documents its participation in the killing of intact hepatocytes by H2O2. Cumene hydroperoxide but not t-butyl hydroperoxide closely models the toxicity of hydrogen peroxide.