Jean-Marc Moulis

High-yield chemical assembly of [2Fe-2X] (X = S, Se) clusters into spinach apoferredoxin: product characterization by resonance Raman spectroscopy

Abstract Conditions are described for the non-enzymic assembly of [2Fe-2S] or [2Fe-2Se] chromophores into spinach apoferredoxin with high yields (60% and 40%, respectively) under physiologically relevant conditions. The success of these reconstitution reactions was found to be critically dependent on the conditions used for the denaturation of native ferredoxin (0.5 M HCl, anaerobic conditions). Low-temperature resonance Raman spectra of native and Se-substituted spinach ferredoxin have been recorded and compared. Most spectral features are shifted to lower frequencies upon S ∗ → Se ∗ substitution, due to the larger atomic mass of selenium compared to sulfur. As a result, each of the spectra displays characteristic bands which are absent in the other. This observation has been used to show that some preparations of Se-substituted ferredoxin also contain [2Fe-2S] chromophores, most probably arising from residual inorganic sulfur bound to apoprotein prepared under non-optimal conditions. Quantitative estimations have shown that the presence of a few percent of residual sulfur in Se-substituted ferredoxin can be detected by resonance Raman spectroscopy. This technique has in addition been used to demonstrate that ferredoxin preparations containing both chalcogenides involve hybrid [2Fe-S-Se] clusters in addition to the [2Fe-2S] and [2Fe-2Se] ones.

Design and functional expression in Escherichia coli of a synthetic gene encoding Clostridium pasteurianum 2[4Fe-4S] ferredoxin

A gene encoding the exact sequence of Clostridium pasteurianum 2[4Fe-4S] ferredoxin and containing 11 unique restriction endonuclease cleavage sites has been synthesized and cloned in Escherichia coli. The synthetic gene is efficiently expressed in E. coli and its product has been purified and characterized. The N-terminal sequence is identical to that of the protein isolated from C. pasteurianum and the recombinant ferredoxin contains the exact amount of [4Fe-4S] clusters (2 per monomer) expected for homogeneous holoferredoxin. It displays reduction potential and kinetic parameters as electron donor to C. pasteurianum hydrogenase I identical to those determined for the native ferredoxin. All of these properties demonstrate that the 2[4Fe-4S] ferredoxin expressed in E. coli is identical to the parent clostridial protein.

Replacement of sulfide by selenide in the [4Fe-4S] clusters of the ferredoxin fromClostridium pasteurianum

Abstract The sulfur atoms of the two [4Fe-4S] clusters present in the ferredoxin from C. pasteurianum have been replaced by selenium. The optical absorption spectrum of the Se-ferredoxin is slightly different from the spectrum of the native protein, but it displays the characteristic features of [4Fe-4X] ( X = S, Se) clustors. The reduced Se-ferredoxin can reduce hydrogenase, and the oxidized Se-ferredoxin can be reduced by hydrogenase in the presence of molecular hydrogen. This is the first report of sulfide substitution by selenide in an iron-sulfur protein containing [4Fe-4S] active sites.

Resonance Raman spectroscopy of [2Fe−2X]2+ (X = S, Se) clusters in ferredoxins

Abstract Low-temperature (approx. 20 K) resonance Raman spectra of native Clostridium pasteurianum (2Fe-2S) ferredoxin, native spinach ferredoxin and Se-substituted spinach ferrodoxin have been obtained with various excitation wavelengths in the 350–500 nm range. All three proteins display at least seven bands in the frequency range where Fe-X stretching modes are expected. 32 S ∗ → 34 S ∗ isotopic substitution on the core sulfide atoms of spinach ferrodoxin results in nearly uniform relative shifts of all bands observed in the 250–450 cm −1 range, presumably because of extensive coupling between the bridging and the terminal stretching modes of the chromophore. However, in the case of Se-substituted spinach ferrodoxin, 76 Se ∗ → 82 Se ∗ isotopic substitution allows discrimination between these two types of modes. Depolarization ratios of the Raman bands of the three proteins have been measured on frozen solutions, using ammonium sulfate as an internal standard. All three proteins display at least four, and most probably six, totally symmetric Fe-X stretching modes. It is inferred from this that the [2Fe-2X]Scys 4 chromophores experience distortions lowering their symmetry point group from D 2h to C 2v , C 2 , or more likely to C S . The present resonance Raman spectra of spinach ferredoxin and of C. pasteurianum [2Fe-2S] ferredoxin have been compared with the previously reported ones of adrenodoxin.

Iron for proliferation of cell lines and hematopoietic progenitors: Nailing down the intracellular functional iron concentration.

Iron is an essential nutrient which must be provided in sufficient amounts to support growth of eukaryotic cells. All organisms devote specialized pathways to ensure proper delivery. Yet, a quantitative assessment of the intra-cellular iron concentration needed to allow the cell cycle to proceed in mammalian cells is missing. Starting from iron-depleted cell lines or primary hematopoietic progenitors prepared with clinically implemented iron chelators, replenishment via transferrin and other iron sources has been quantitatively monitored through the main endogenous markers of the cellular iron status, namely proteins involved in the uptake (transferrin receptor), the storage (ferritin), and the sensing (Iron Regulatory Proteins) of iron. When correlated with measurements of iron concentrations and indicators of growth, this minimally intrusive approach provided an unprecedented estimate of the intracellular iron concentration acting upon iron-centered regulatory pathways. The data were analyzed with the help of a previously developed theoretical treatment of cellular iron regulation. The minimal cellular iron concentration required for cell division was named functional iron concentration (FIC) to distinguish it from previous estimates of the cellular labile iron. The FIC falls in the low nanomolar range for all studied cells, including hematopoietic progenitors. These data shed new light on basic aspects of cellular iron homeostasis by demonstrating that sensing and regulation of iron occur well below the concentrations requiring storage or becoming noxious in pathological conditions. The quantitative assessment provided here is relevant for monitoring treatments of conditions in which iron provision must be controlled to avoid unwanted cellular proliferation.

Resonance Raman spectroscopy of Azotobacter vinelandii ferredoxin I: Vibrational features of the [3Fe-3S] cluster

Low temperature resonance Raman spectra have been obtained for Clostridium pasteurianum and Bacillus stearothermophilus ferredoxins. Several heretofore undetected fundamental bands have been observed and these data have been used to discriminate the vibrational contribution of the [3Fe‐3S] cluster to the spectrum of Azotobacter vinelandii ferredoxin I. The vibrational features of the [3Fe‐3S] core distinguish it from other 3‐iron clusters and imply structural differences among this class of iron‐sulfur clusters.

Chronic Exposure to Low-Level Cadmium in Diabetes: Role of Oxidative Stress and Comparison with Polychlorinated Biphenyls

Among the most important physiological functions, maintenance of the oxidation reduction equilibrium in cells stands out as a major homeostatic event. Many environmental contaminants efficiently trap cellular reducing compounds, but the actual importance of this mode of toxicity is far from being precisely known. This statement applies to cases of slowly developing chronic diseases, such as neurodegenerations, diabetes, and many others. The involvement of oxidative challenge in diabetes is considered in connection with chronic dietary exposure to low-level concentrations of cadmium. Comparison is made with polychlorobiphenyl molecules (PCB): they are structurally unrelated to cadmium, they preferentially distribute into different organs than cadmium, and they follow different metabolic pathways. Yet, they have also pro-oxidative properties, and they are associated with diabetes. Since neither cadmium nor PCB is a direct oxidant, they both follow indirect pathways to shift the redox equilibrium. Thus, a difference must be made between the adaptable response of the organism, i.e. the anti-oxidant response, and the irreversible damage generated by oxidizing species, i.e. oxidative damage, when exposure occurs at low concentrations. The approximate border between high and low levels of exposure is estimated in this review from the available relevant data, and the strengths and weaknesses of experimental models are delineated. Eventually, chronic low level exposure to these contaminants sparks cellular responses setting ground for dysfunction and disease, such as diabetes: oxidative damage is an accompanying phenomenon and not necessarily an early mechanism of toxicity.

Mitochondrial Morphology and Function of the Pancreatic β-Cells INS-1 Model upon Chronic Exposure to Sub-Lethal Cadmium Doses

The impact of chronic cadmium exposure and slow accumulation on the occurrence and development of diabetes is controversial for human populations. Islets of Langerhans play a prominent role in the etiology of the disease, including by their ability to secrete insulin. Conversion of glucose increase into insulin secretion involves mitochondria. A rat model of pancreatic β-cells was exposed to largely sub-lethal levels of cadmium cations applied for the longest possible time. Cadmium entered cells at concentrations far below those inducing cell death and accumulated by factors reaching several hundred folds the basal level. The mitochondria reorganized in response to the challenge by favoring fission as measured by increased circularity at cadmium levels already ten-fold below the median lethal dose. However, the energy charge and respiratory flux devoted to adenosine triphosphate synthesis were only affected at the onset of cellular death. The present data indicate that mitochondria participate in the adaptation of β-cells to even a moderate cadmium burden without losing functionality, but their impairment in the long run may contribute to cellular dysfunction, when viability and β-cells mass are affected as observed in diabetes.

Iron and Oxidative Stress in Gestational Diabetes

Gestational Diabetes mellitus (GDM) is a significantly growing problem in worldwide healthcare, and it is one of the most common pregnancy complications associated with increased perinatal and long‐term adverse effects for both mothers and newborns. Although sufficient iron stores during pregnancy are essential to prevent negative outcomes for both infants and mothers, excess iron increases the risk of GDM. Accumulating data suggest that a high iron intake might increase the risk of GDM and of Type 2 diabetes. Moderately elevated serum ferritin early in pregnancy should be considered as a predictive factor for GDM, and routine evaluation of ferritin levels and other parameters of iron homeostasis should guide supplementation and help identifying and preventing GDM In high‐risk women by providing them with dietary advice.

Impact of chronic and low cadmium exposure of rats: sex specific disruption of glucose metabolism

BACKGROUND Several epidemiological and animal studies suggest a positive association between cadmium (Cd) exposure and incidence of type 2 diabetes, but the association remains controversial. Besides, the experimental data have mainly been obtained with relatively high levels of Cd, over various periods of time, and with artificial routes of administration. OBJECTIVES Do environmental exposures to Cd induce significant disruption of glucose metabolism? METHODS Adults Wistar rats were exposed for three months to 0, 5, 50 or 500 μg.kg-1.d-1 of CdCl2 in drinking water. Relevant parameters of glucose homeostasis were measured. RESULTS Cd accumulated in plasma, kidney and liver of rats exposed to 50 and 500 μg.kg-1.d-1, without inducing signs of organ failure. In rats drinking 5 μg.kg-1.d-1 for 3 months, Cd exposure did not lead to any significant increase of Cd in these organs. At 50 and 500 μg.kg-1.d-1 of Cd, glucose and insulin tolerance were unchanged in both sexes. However, females exhibited a significant increase of both fasting and glucose-stimulated plasma insulin that was assigned to impaired hepatic insulin extraction as indicated by unaltered fasting C-peptide plasma levels. CONCLUSIONS Glucose homeostasis is sensitive to chronic Cd exposure in a gender-specific way. Moreover, this study proves that an environmental pollutant such as Cd can have, at low concentrations, an impact on the glucose homeostatic system and it highlights the importance of a closer scrutiny of the underlying environmental causes to understand the increased incidence of type 2 diabetes.