On CO Egress from,and Re‐Uptake by,the Enzyme MauG,as a Mimic of the Acquisition of Oxidizing Agents by the pre‐MADH_MauG System. A Molecular Mechanics Approach

In this work, by applying a non‐deterministic, randomly‐oriented minimal force to the dissociated CO ligand of the MauG‐CO system, the molecular‐dynamics (MD) behavior of this system could be quickly unraveled. It turned out that CO has no marked directional egress from the high‐spin c‐heme iron distal pocket. Rather, CO is able to exploit all interstices created during the protein fluctuations. Nonetheless, no steady route toward the surrounding solvent was ever observed: CO jumped first into other binding pockets before being able to escape the protein. In a few cases, on hitting the surrounding H₂O molecules, CO was observed to reverse direction, re‐entering the protein. A contention that conformational inversion of the P107 ring provides a gate to the iron ion is not supported by the present simulations.     

The effect of CO2 availability on the growth, iron oxidation and CO2-fixation rates of pure cultures of Leptospirillum ferriphilum and Acidithiobacillus ferrooxidans

Understanding how bioleaching systems respond to the availability of CO(2) is essential to developing operating conditions that select for optimum microbial performance. Therefore, the effect of inlet gas and associated dissolved CO(2) concentration on the growth, iron oxidation and CO(2) -fixation rates of pure cultures of Acidithiobacillus ferrooxidans and Leptospirillum ferriphilum was investigated in a batch stirred tank system. The minimum inlet CO(2) concentrations required to promote the growth of At. ferrooxidans and L. ferriphilum were 25 and 70 ppm, respectively, and corresponded to dissolved CO(2) concentrations of 0.71 and 1.57 μM (at 30°C and 37°C, respectively). An actively growing culture of L. ferriphilum was able to maintain growth at inlet CO(2) concentrations less than 30 ppm (0.31-0.45 μM in solution). The highest total new cell production and maximum specific growth rates from the stationary phase inocula were observed with CO(2) inlet concentrations less than that of air. In contrast, the amount of CO(2) fixed per new cell produced increased with increasing inlet CO(2) concentrations above 100 ppm. Where inlet gas CO(2) concentrations were increased above that of air the additional CO(2) was consumed by the organisms but did not lead to increased cell production or significantly increase performance in terms of iron oxidation. It is proposed that At. ferrooxidans has two CO(2) uptake mechanisms, a high affinity system operating at low available CO(2) concentrations, which is subject to substrate inhibition and a low affinity system operating at higher available CO(2) concentrations. L. ferriphilum has a single uptake system characterised by a moderate CO(2) affinity. At. ferrooxidans performed better than L. ferriphilum at lower CO(2) availabilities, and was less affected by CO(2) starvation. Finally, the results demonstrate the limitations of using CO(2) uptake or ferrous iron oxidation data as indirect measures of cell growth and performance across varying physiological conditions.     

Regulation of the 75-kDa subunit of mitochondrial complex I by iron

Iron homeostasis is tightly regulated, as cells work to conserve this essential but potentially toxic metal. The translation of many iron proteins is controlled by the binding of two cytoplasmic proteins, iron regulatory protein 1 and 2 (IRP1 and IRP2) to stem loop structures, known as iron-responsive elements (IREs), found in the untranslated regions of their mRNAs. In short, when iron is depleted, IRP1 or IRP2 bind IREs; this decreases the synthesis of proteins involved in iron storage and mitochondrial metabolism (e.g. ferritin and mitochondrial aconitase) and increases the synthesis of those involved in iron uptake (e.g. transferrin receptor). It is likely that more iron-containing proteins have IREs and that other IRPs may exist. One obvious place to search is in Complex I of the mitochondrial respiratory chain, which contains at least 6 iron-sulfur (Fe-S) subunits. Interestingly, in idiopathic Parkinson's disease, iron homeostasis is altered, and Complex I activity is diminished. These findings led us to investigate whether iron status affects the Fe-S subunits of Complex I. We found that the protein levels of the 75-kDa subunit of Complex I were modulated by levels of iron in the cell, whereas mRNA levels were minimally changed. Isolation of a clone of the 75-kDa Fe-S subunit with a more complete 5'-untranslated region sequence revealed a novel IRE-like stem loop sequence. RNA-protein gel shift assays demonstrated that a specific cytoplasmic protein bound the novel IRE and that the binding of the protein was affected by iron status. Western blot analysis and supershift assays showed that this cytosolic protein is neither IRP1 nor IRP2. In addition, ferritin IRE was able to compete for binding with this putative IRP. These results suggest that the 75-kDa Fe-S subunit of mitochondrial Complex I may be regulated by a novel IRE-IRP system.     

High affinity iron-uptake systems in Vibrio damsela: role in the acquisition of iron from transferrin

In this work, the high affinity iron-acquisition systems displayed by virulent and avirulent strains of Vibrio damsela have been investigated. This species is an autochthonous member of marine ecosystems that can behave as an opportunistic pathogen for fish and mammals. All strains tested (i) were able to grow under the restricted conditions imposed by the iron chelators transferrin (Tf) and EDDHA, (ii) secreted siderophores of hydroxamic type, other than aerobactin and desferal, that were able to stimulate the growth of the auxotroph mutant Arthrobacter flavescens JG9, and (iii) expressed common iron-regulated outer membrane proteins (IROMPs). No change in LPS patterns was observed in response to iron restriction. Results from the assays with transferrin suggest that these siderophores could be utilized to sequester iron from Tf, a protein for which no surface receptor was detected in any strain. In summary, the overall data demonstrate that V. damsela expresses siderophore-mediated iron-uptake systems. These systems are probably involved in the survival of the species in the different environments that it can colonize, i.e. water and several vertebrate hosts.     

Carbon monoxide improves adaptation of Arabidopsis to iron deficiency

Carbon monoxide (CO) is an endogenous gaseous molecule and regulates a variety of biological processes in animals. However, whether CO regulates nutrient stress responses in plants is largely unknown. In this paper, we described an observation that CO can regulate iron-homeostasis in iron-starved Arabidopsis. Exogenous CO at 50 μ m was able to prevent the iron deficient-induced chlorosis and improve chlorophyll accumulation. Expression of AtIRT1 , AtFRO2 , AtFIT1 and AtFER1 was up-regulated by CO exposure in iron-deficient seedlings. CO-regulated iron homeostasis could also be found in monocot maize and green alga Chlamydomonas reinhardtii . Treatment with external CO increased iron accumulation in iron-deficient Arabidopsis and C. reinhardtii , and restored leaf greening in Maize ys1 and ys3 mutants (defective in Fe uptake). Moreover, endogenous CO level was increased in Arabidopsis under iron-deficiency. Finally, CO exposure induced NO accumulation in root tips. However, such an action could be blocked by NO scavenger cPTIO. These results indicate that CO may play an important role in improving plant adaptation to iron deficiency or cross-talking with NO under the iron deficiency.     

Characterization and regulation of the expression of FatB, an iron transport protein encoded by the pJM1 virulence plasmid

The pJM1-encoded genes fatDCBA are essential for iron acquisition via the siderophore anguibactin. Sequence analysis indicated that the open reading frame corresponding to the fatB gene possesses domains that are characteristic of periplasmic proteins that bind the ferric siderophore. In this work, a monospecific antiserum against an oligopeptide containing the last 27 amino acids of the carboxy-terminal region from this open reading frame was used to demonstrate that fatB encodes a 35 kDa protein that is essential for iron transport. By using this antibody we were able to demonstrate that expression of the fatB gene is negatively regulated by the Fur protein at high iron concentrations. Conversely, its expression was positively regulated by the combined action of the AngR protein and products of the TAF region. FatB, the product of the fatB gene, is isolated with the membrane fraction. In accordance with these findings is the fact that the first 23 amino acid residues of this protein have the properties of a lipoprotein signal sequence. The lipoprotein nature of FatB is supported by the fact that treatment of Vibrio anguillarum cells with globomycin, an inhibitor of the lipoprotein signal peptidase, results in the accumulation of a 38 kDa pro-FatB precursor protein.     

Alteration of heme axial ligands in hemoglobin by organic solvents analyzed by CD, FTIR, and XANES techniques

The effects of mixed solvents on the ligand binding site in hemoglobin have been investigated though three spectroscopic techniques. Two classes of organic solvents (amides and alcohols) known to increase or decrease the hemoglobin affinity have been chosen for this study. The analysis of the iron CO stretching band shows that the ligand binding sites of alpha CO and beta CO subunits inside the alpha 2 beta 2 hemoglobin tetramer exhibit multiple conformations. From the circular dichroism and X-ray absorption near-edge structure data, it appears that no core deformation or heme reorientation occur with the affinity changes. The iron-ligand average bond angle is the sole parameter that depends on the external solvent. Since cosolvents seem to affect the dynamics rather than the hindrance of the heme cavity, we suggest that the protein affinity could be associated with a hierarchy of subtle dynamic states.     

Exochelin-mediated iron acquisition by the leprosy bacillus, Mycobacterium leprae

Exochelins, water-soluble siderophores of mycobacteria, were isolated and partially purified from culture filtrates of iron-deficiently grown cultures of Mycobacterium neoaurum NCTC 10439 and an armadillo-derived Mycobacterium (ADM 8563). Two biologically active fractions mediating iron uptake were isolated from each bacterium which not only were able to transport iron into the producing organism but also into suspensions of Mycobacterium leprae isolated from armadillo liver. The rate of exochelin-mediated iron uptake into M. leprae was about 1.5% of the rate observed into the producing organisms. The process of iron uptake appears to be by facilitated diffusion as it was not inhibited by HgCl2, NaN3, KCN, dinitrophenol or carbonyl cyanide m-chlorophenylhydrazone. Since no uptake of iron occurred into iron-sufficient ADM cells, this may indicate that M. leprae, as recovered from an animal tissue, had been growing iron-deficiently in order for iron uptake to have been demonstrated in vitro.     

Purification and characterization of carbon monoxide dehydrogenase, a nickel, zinc, iron-sulfur protein, from Rhodospirillum rubrum

Carbon monoxide dehydrogenase (CO dehydrogenase) from Rhodospirillum rubrum was shown to be an oxygen-sensitive, nickel, iron-sulfur, and zinc-containing protein that was induced by carbon monoxide (CO). The enzyme was purified 212-fold by heat treatment, ion-exchange, and hydroxylapatite chromatography and preparative gel electrophoresis. The purified protein, active as a monomer of Mr = 61,800, existed in two forms that were comprised of identical polypeptides and differed in metal content. Form 1 comprised 90% of the final activity, had a specific activity of 1,079 mumol CO oxidized per min-1 mg-1, and contained 7 iron, 6 sulfur, 0.6 nickel, and 0.4 zinc/monomer. Form 2 had a lower specific activity (694 mumol CO min-1 mg-1) and contained 9 iron, 8 sulfur, 1.4 nickel, and 0.8 zinc/monomer. Reduction of either form by CO or dithionite resulted in identical, rhombic ESR spectra with g-values of 2.042, 1.939, and 1.888. Form 2 exhibited a 2-fold higher integrated spin concentration, supporting the conclusion that it contained an additional reducible metal center(s). Cells grown in the presence of 63NiCl2 incorporated 63Ni into CO dehydrogenase. Although nickel was clearly present in the protein, it was not ESR-active under any conditions tested. R. rubrum CO dehydrogenase was antigenically distinct from the CO dehydrogenases from Methanosarcina barkeri and Clostridium thermoaceticum.     

Oxidation-induced ferritin turnover in microglial cells: role of proteasome

Highly oxidized protein aggregates accumulating in the brain during neurodegenerative diseases are often surrounded by microglia. Most of the microglial cells surrounding these plaques are activated and release a high amount of oxidizing species. In order to develop their toxic effects numerous oxidizing species need iron. To prevent this iron-dependent oxidation an iron-sequestering apparatus exists, including the major iron storage protein ferritin. Microglial cells damage their own protein pool during activation and it is still unknown whether microglial cells are able to maintain their iron-sequestering function during oxidative stress. Therefore, we explored the microglial cell line RAW to test the maintenance of ferritin under oxidizing conditions. Our investigations revealed a half-life of both ferritin chains of 3-3.5 h and a reduced half-life due to oxidation. This was due to the removal of oxidized ferritin by the proteasomal system. Ferritin de novo synthesis was also severely affected by oxidation. This results in a decreased ferritin pool due to acute oxidative stress. These data let us conclude that microglial cells do not increase their ferritin amount after oxidative stress and an increase in the iron storage capacity in these cells after treatment might be achieved only by a high iron saturation of the existing ferritin molecules.     

Effect of Glucose on Carbon Dioxide Assimilation and Substrate Oxidation by Ferrobacillus ferrooxidans

Ferrobacillus ferrooxidans, grown on either elemental sulfur or ferrous sulfate, was able to use either substrate as an energy source for the assimilation of CO(2). In both cases, 0.01 mumole of carbon was incorporated per mumole of oxygen utilized. Glucose inhibited substrate oxidation and CO(2) fixation. Sulfur and iron oxidation were inhibited 5 to 15% and 40 to 50%, respectively, in the presence of 10% glucose. Under the same conditions, CO(2) assimilation was inhibited 50% with elemental sulfur as the energy source, and was almost totally inhibited when ferrous iron was used.     

Gene Regulation of Iron-Deficiency Responses Is Associated with Carbon Monoxide and Heme Oxydase 1 in Chlamydomonas reinhardtii

Carbon monoxide (CO) as an endogenous gaseous molecule regulates a variety of biological processes in animals. However, CO regulating nutrient stress responses in green alga is largely unknown. On the other hand, heme oxydase (HO1 as a rate-limiting enzyme of the first step for heme degration and to catalyze heme into biliverdin (BV), which is concomitant with releasing of CO and ferrous ions, probably participates in the process of CO-regulating response to nutrient stress in green alga. In this paper, we described an observation that CO could regulate iron-homeostasis in iron-starving Chlamydomonas reinhardtii. Exogenous CO at 8 µM was able to prevent the iron deficient-inducing chlorosis and improve chlorophyll accumulation. Expression pattern of FOX1, FTR1 and ferredoxin was up-regulated by CO exposure in iron-deficient mediam. treatment with external CO increasing iron accumulation in iron-deficient C. reinhardtii. Moreover, to get insights into the regulatory role of HO1, we constructed a transgenic alga overexpressing HO1 and HO1 knock-out mutants. The results show that there was no significant influence on chlorosis with HO1 overexpression of C. reinhardtii under iron-deficiency and the chlorophyll accumulation, and gene expression associated with iron deficiency of mutant were greatly improved. Otherwise, those results from HO1 knock-out mutants were opposite to HO1 overexpression mutants. Finally, CO exposure induced NO accumulation in cells. However, such an action could be blocked by NO scavenger cPTIO. These results indicate that CO/HO1 may play an important role in improving green algae adaptation to iron deficiency or cross-talking with NO under the iron deficiency.     

The incorporation of iron into apoferritin as mediated by ceruloplasmin

Ceruloplasmin, a copper ferroxidase, promotes the incorporation of Fe(III) into the iron storage protein, apoferritin. The product formed is identical to ferritin as judged by polyacrylamide electrophoresis and iron/protein measurements. Of several proteins examined, only apoferritin accumulates the Fe(III) produced by ceruloplasmin. When ceruloplasmin was replaced by tyrosinase, which we have shown to have ferroxidase activity, no iron incorporation into apoferritin was observed. It is proposed that Fe(III) is transferred directly and specifically to apoferritin. These data support a more specific role for ceruloplasmin in iron metabolism than has previously been proposed.     

Utilization of hemin and hemoglobin by Vibrio vulnificus biotype 2.

The eel pathogen Vibrio vulnificus biotype 2 is able to use hemoglobin (Hb) and hemin (Hm) to reverse iron limitation. In this stud, the adjuvant effect of both compounds on eel pathogenicity has been evaluated and confirmed. Further, we have studied the heme-iron acquisition mechanism displayed by this bacterium. Whole cells were capable of binding Hb and Hm, independently of (i) iron levels in growth medium and (ii) the presence of polysaccharide capsules on bacterial surface. The Hb- and Hm-binding capacity was retained by the outer membrane protein (OMP) fraction and was abolished after proteolytic digestion of OMP samples. Western blotting (immunoblotting) of denatured OMPs revealed that two major protein bands of 36 and 32 kDa were involved in both Hm and Hb binding. The expression of these proteins was not affected by iron levels. In addition, V. vulnificus biotype 2 produced extracellular proteases, not regulated by iron, that were active against native Hb. In conclusion, the overall data suggest that the eel pathogen V. vulnificus biotype 2 can obtain iron by means of a mechanism which involves a direct interaction between the heme moiety and constitutive OMPs.     

Iron binding proteins and influx of iron across the duodenal brush border. Evidence for specific lactotransferrin receptors in the human intestine.

The ability of a range of homologous transferrin-like proteins to donate iron to pieces of human duodenal mucosa, was examined with an in vitro incubation technique. In contrast to serum transferrin and ovotransferrin, only lactotransferrin was able to yield its iron to intestinal tissue, but in an autologous system this protein was unable to donate iron to human reticulocyte preparations. Studies with 125I-labelled lactotransferrin and lactotransferrin dual-labelled with 59Fe and 125I, indicated that the intact protein is excluded from entry into the enterocytes. The experiments suggest that iron may be transported across the brush border after delivery to specific protein binding sites at the cell surface.