The two iron regulatory proteins IRP1 and IRP2 bind to transcripts of ferritin, transferrin receptor and other target genes to control the expression of iron metabolism proteins at the post-transcriptional level. Here we compare the effects of genetic ablation of IRP1 to IRP2 in mice. IRP1À/À mice misregulate iron metabolism only in the kidney and brown fat, two tissues in which the endogenous expression level of IRP1 greatly exceeds that of IRP2, whereas IRP2À/À mice misregulate the expression of target proteins in all tissues. Surprisingly, the RNA-binding activity of IRP1 does not increase in animals on a lowiron diet that is sufficient to activate IRP2. In animal tissues, most of the bifunctional IRP1 is in the form of cytosolic aconitase rather than an RNA-binding protein.Our findings indicate that the small RNA-binding fraction of IRP1, which is insensitive to cellular iron status, contributes to basal mammalian iron homeostasis, whereas IRP2 is sensitive to iron status and can compensate for the loss of IRP1 by increasing its binding activity. Thus, IRP2 dominates post-transcriptional regulation of iron metabolism in mammals.

Fe-only hydrogenases contain a di-iron active site complex, in which the two Fe atoms have carbon monoxide and cyanide ligands and are linked together by a putative di(thiomethyl)amine molecule. We have cloned, purified and characterized the HydE and HydG proteins, thought to be involved in the biosynthesis of this peculiar metal site, from the thermophilic organism Thermotoga maritima. The HydE protein anaerobically reconstituted with iron and sulfide binds two [4Fe-4S] clusters, as characterized by UV and EPR spectroscopy. The HydG protein binds one [4Fe-4S] cluster, and probably an additional one. Both enzymes are able to reductively cleave S-adenosylmethionine (SAM) when reduced by dithionite, confirming that they are Radical-SAM enzymes.

Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) hydrolyze the neurotransmitter acetylcholine and, thereby, function as coregulators of cholinergic neurotransmission. Although closely related, these enzymes display very different substrate specificities that only partially overlap. This disparity is largely due to differences in the number of aromatic residues lining the active site gorge, which leads to large differences in the shape of the gorge and potentially to distinct interactions with an individual ligand. Considerable structural information is available for the binding of a wide diversity of ligands to AChE. In contrast, structural data on the binding of reversible ligands to BChE are lacking. In a recent effort, an inhibitor competition approach was used to probe the overlap of ligand binding sites in BChE. Here, we extend this study by solving the crystal structures of human BChE in complex with five reversible ligands, namely, decamethonium, thioflavin T, propidium, huprine, and ethopropazine. We compare these structures to equivalent AChE complexes when available in the protein data bank and supplement this comparison with kinetic data and observations from isothermal titration calorimetry. This new information now allows us to define the binding mode of various ligand families and will be of importance in designing specific reversible ligands of BChE that behave as inhibitors or reactivators.

The active site of [Fe-Fe]-hydrogenases is composed of a di-iron complex, where the two metal atoms are bridged together by a putative di(thiomethyl)amine molecule and are also ligated by di-nuclear ligands, namely carbon monoxide and cyanide. Biosynthesis of this metal site is thought to require specific protein machinery coded by the hydE, hydF, and hydG genes. The HydF protein has been cloned from the thermophilic organism Thermotoga maritima, purified, and characterized. The enzyme possesses specific amino acid signatures for GTP binding and is able to hydrolyze GTP. The anaerobically reconstituted TmHydF protein binds a [4Fe-4S] cluster with peculiar EPR characteristics: an S ‫؍‬ 1/2 signal presenting a high field shifted g-value together with a S ‫؍‬ 3/2 signal, similar to those observed for [4Fe-4S] clusters ligated by only three cysteines. HYSCORE spectroscopy experiments were carried out to determine the nature of the fourth ligand of the cluster, and its exchangeability was demonstrated with the formation of a [4Fe-4S]-imidazole complex.Hydrogenases are metalloproteins that catalyze the reversible activation of molecular hydrogen and enable an organism to either utilize H 2 as a source of reducing power or to use protons as terminal electron acceptors, thus generating H 2 gas. Based on their metal content, hydrogenases are divided into three classes, [Ni-Fe]-hydrogenases (1), [FeFe]-hydrogenases (2, 3) and "iron-sulfur cluster-free" hydrogenase (4 -6), which do not appear to be structurally or phylogenetically related (7).[Fe-Fe]-hydrogenases are limited to certain anaerobic bacteria and anaerobic eukaryotes and are often involved in H 2 evolution with catalytic activities up to 100 times higher than those of [Ni-Fe]-hydrogenases (8). At their active site they contain a dinuclear iron center attached to the protein by only one bond between a cysteine residue and one of the two iron atoms (Fig. 1). This cysteine also serves as a ligand for an adjacent [4Fe-4S] cluster, so there is a sulfur bridge between the two metal sites (2, 3). [Fe-Fe]-hydrogenases also contain additional [2Fe-2S] and [4Fe-4S] clusters, which shuttle electrons between the H 2 activating site, inside the protein, and the redox partners at the surface.One remarkable property of the [Ni-Fe]-and [Fe-Fe]-hydrogenase active sites is the presence of carbon monoxide and cyanide ligands as clearly established by x-ray crystallography (1-3) and infrared spectroscopy (9). In both cases CO and CN Ϫ are found coordinated to the iron atoms and are thought to allow stabilization of the low iron oxidation and spin states required for activity (10). Infrared spectroscopy studies have also demonstrated the presence of CO ligands in the "iron-sulfur cluster-free" hydrogenase (11). In the unique case of [Fe-Fe]-hydrogenases an intriguing low molecular weight compound, still incompletely identified but often proposed to be a di(thiomethyl)amine, is bound to the di-iron site through a bridging bidentate coordination mode ( Fig. 1) (12). The presence of p...