Fe2+ binds iron responsive element-RNA, selectively changing protein-binding affinities and regulating mRNA repression and activation.

Iron increases synthesis rates of proteins encoded in iron-responsive element (IRE)-mRNAs; metabolic iron ("free," "labile") is Fe(2+). The noncoding IRE-RNA structure, approximately 30 nt, folds into a stem loop to control synthesis of proteins in iron trafficking, cell cycling, and nervous system function. IRE-RNA riboregulators bind specifically to iron-regulatory proteins (IRP) proteins, inhibiting ribosome binding. Deletion of the IRE-RNA from an mRNA decreases both IRP binding and IRP-independent protein synthesis, indicating effects of other "factors." Current models of IRE-mRNA regulation, emphasizing iron-dependent degradation/modification of IRP, lack answers about how iron increases IRE-RNA/IRP protein dissociation or how IRE-RNA, after IRP dissociation, influences protein synthesis rates. However, we observed Fe(2+) (anaerobic) or Mn(2+) selectively increase the IRE-RNA/IRP K(D). Here we show: (i) Fe(2+) binds to the IRE-RNA, altering its conformation (by 2-aminopurine fluorescence and ethidium bromide displacement); (ii) metal ions increase translation of IRE-mRNA in vitro; (iii) eukaryotic initiation factor (eIF)4F binds specifically with high affinity to IRE-RNA; (iv) Fe(2+) increased eIF4F/IRE-RNA binding, which outcompetes IRP binding; (v) exogenous eIF4F rescued metal-dependent IRE-RNA translation in eIF4F-depeleted extracts. The regulation by metabolic iron binding to IRE-RNA to decrease inhibitor protein (IRP) binding and increase activator protein (eIF4F) binding identifies IRE-RNA as a riboregulator.

Oxidative stress-related genes in type 2 diabetes: association analysis and their clinical impact.

Worldwide prevalence of diabetes mellitus motivates a number of association studies to be conducted throughout the world. Eleven polymorphisms from nine candidate genes in oxidative stress pathway have been analyzed in eastern Indian type 2 diabetic patients (n = 145) and healthy controls (n = 100). Different biochemical parameters were also analyzed for their association with the disease. Significant associations were observed for rs2070424 A>G SOD1 (OR 3.91, 95% CI 2.265-8.142, P < 0.001), rs854573 A>G PON1 (OR 3.415, 95% CI 2.116-5.512, P < 0.001), rs6954345 G>C PON2 (OR 3.208, 95% CI 2.071-4.969, P < 0.001), RAGE rs1800624 -374 T>A (OR 3.58, 95% CI 2.218-5.766, P < 0.001), and NOS3 -786 T>C (OR 3.75, 95% CI 2.225-6.666, P < 0.001). Haplotype containing two risk alleles of PON1 and PON2 genes was significantly associated with disease (OR 8.34, 95% CI 1.554-44.804, P < 0.002). Our results suggest that carriers of major and efficient alleles of oxidative stress genes are more likely to survive the comorbid complications and single copy of risk allele is sufficient for developing the disease.

Spectroscopic studies of single and double variants of M ferritin: lack of conversion of a biferrous substrate site into a cofactor site for O2 activation.

Ferritin has a binuclear non-heme iron active site that functions to oxidize iron as a substrate for formation of an iron mineral core. Other enzymes of this class have tightly bound diiron cofactor sites that activate O2 to react with substrate. Ferritin has an active site ligand set with 1-His/4-carboxylate/1-Gln rather than the 2-His/4-carboxylate set of the cofactor site. This ligand variation has been thought to make a major contribution to this biferrous substrate rather than cofactor site reactivity. However, the Q137E/D140H double variant of M ferritin, has a ligand set that is equivalent to most of the diiron cofactor sites, yet did not rapidly react with O2 or generate the peroxy intermediate observed in the cofactor sites. Therefore, in this study, a combined spectroscopic methodology of circular dichroism (CD)/magnetic CD (MCD)/variable temperature, variable field (VTVH) MCD has been applied to evaluate the factors required for the rapid O2 activation observed in cofactor sites. This methodology defines the coordination environment of each iron and the bridging ligation of the biferrous active sites in the double and corresponding single variants of frog M ferritin. Based on spectral changes, the D140H single variant has the new His ligand binding, and the Q137E variant has the new carboxylate forming a µ-1,3 bridge. The spectra for the Q137E/D140H double variant, which has the cofactor ligand set, however, reflects a site that is more coordinately saturated than the cofactor sites in other enzymes including ribonucleotide reductase, indicating the presence of additional water ligation. Correlation of this double variant and the cofactor sites to their O2 reactivities indicates that electrostatic and steric changes in the active site and, in particular, the hydrophobic nature of a cofactor site associated with its second sphere protein environment, make important contributions to the activation of O2 by the binuclear non-heme iron enzymes.

Moving Iron through ferritin protein nanocages depends on residues throughout each four α-helix bundle subunit.

Eukaryotic H ferritins move iron through protein cages to form biologically required, iron mineral concentrates. The biominerals are synthesized during protein-based Fe²âº/O2 oxidoreduction and formation of [Fe³âºO](n) multimers within the protein cage, en route to the cavity, at sites distributed over ~50 Å. Recent NMR and Co²âº-protein x-ray diffraction (XRD) studies identified the entire iron path and new metal-protein interactions: (i) lines of metal ions in 8 Fe²âº ion entry channels with three-way metal distribution points at channel exits and (ii) interior Fe³âºO nucleation channels. To obtain functional information on the newly identified metal-protein interactions, we analyzed effects of amino acid substitution on formation of the earliest catalytic intermediate (diferric peroxo-A(650 nm)) and on mineral growth (Fe³âºO-A(350 nm)), in A26S, V42G, D127A, E130A, and T149C. The results show that all of the residues influenced catalysis significantly (p < 0.01), with effects on four functions: (i) Fe²âº access/selectivity to the active sites (Glu¹³°), (ii) distribution of Fe²âº to each of the three active sites near each ion channel (Asp¹²7), (iii) product (diferric oxo) release into the Fe³âºO nucleation channels (Ala²6), and (iv) [Fe³âºO](n) transit through subunits (Val4², Thr¹49). Synthesis of ferritin biominerals depends on residues along the entire length of H subunits from Fe²âº substrate entry at 3-fold cage axes at one subunit end through active sites and nucleation channels, at the other subunit end, inside the cage at 4-fold cage axes. Ferritin subunit-subunit geometry contributes to mineral order and explains the physiological impact of ferritin H and L subunits.

An engineered pathway for the formation of protein disulfide bonds.

We have engineered a pathway for the formation of disulfide bonds. By imposing evolutionary pressure, we isolated mutations that changed thioredoxin, which is a monomeric disulfide reductase, into a [2Fe-2S] bridged dimer capable of catalyzing O2-dependent sulfhydryl oxidation in vitro. Expression of the mutant protein in Escherichia coli with oxidizing cytoplasm and secretion via the Tat pathway restored disulfide bond formation in strains that lacked the complete periplasmic oxidative machinery (DsbA and DsbB). The evolution of [2Fe-2S] thioredoxin illustrates how mutations within an existing scaffold can add a cofactor and markedly change protein function.