The preparation and characterization of a series of encapsulated-lanthanide 15-metallacrown-5 complexes are reported. Planar ligands such as picoline hydroxamic acid (picha) or nonplanar alpha-amino hydroxamic acids (e.g., glycine hydroxamic acid (glyha)) led to one-step syntheses of metallacrowns in yields as high as 85%. The reaction of the appropriate hydroxamic acid with copper acetate and (1)/(5) equiv of gadolinium(III) or europium(III) nitrates in DMF or water yielded crystals of Gd(NO(3))(3)[15-MC(Cu(II)N(picha))-5], 1, Eu(NO(3))(3)[15-MC(Cu(II)N(picha))-5], 2, and Eu(NO(3))(3)[15-MC(Cu(II)N(glyha))-5], 3. Several other 15-metallacrown-5 complexes were synthesized with (1) Cu(II) or Ni(II) in the metallacrown ring metal position, (2) various lanthanides (La(III), Nd(III), Sm(III), Eu(III), Gd(III), Dy(III), Ho(III), Er(III), and Yb(III)) encapsulated in the center of the ring, and (3) chiral alpha-amino hydroxamic acids (e.g., phenylalanine hydroxamic acid (H(2)pheha), leucine hydroxamic acid (H(2)leuha), and tyrosine hydroxamic acid (H(2)tyrha)). It is believed that all of the complexes containing Cu(II) ions have the ring metals either in four-coordinate, square-planar environments, bound to two tetradentate hydroximate ligands, or in five-coordinate, square-pyramidal geometries if solvent is bound. Spectroscopic and magnetic characterization of the Ni(II) complexes suggests that they are either five- or six-coordinate. The encapsulated lanthanides are generally pentagonal bipyramidal, with five oxygen donors from the metallacrown ring and solvent or bidentate nitrate ions in the axial positions. The circular arrangement of ions results in interesting magnetic behavior. With Dy(III) encapsulated in the center of the ring, a magnetic moment as high as 10.9 &mgr;(B) is achieved. Analysis of the variable-temperature susceptibility of La(NO(3))(3)[15-MC(Cu(II)N(picha))-5] indicates that the five Cu(II) ions are antiferromagnetically coupled, forming an S = (1)/(2) ground spin state with a moment of 1.7 &mgr;(B) at liquid helium temperatures. Complex 1 shows ferromagnetic coupling of the Gd(III) ion to the five Cu ions at temperatures below 15 K. Studies of the metallacrown complexes in solution show that they are stable and soluble in DMF and water. A proton relaxation study on complex 1 has revealed a relaxivity of 9.8 mM(-)(1) s(-)(1) (20 degrees C and 30 MHz), a value that is comparable to those of clinically useful MRI contrast enhancement agents. Complex 1 crystallizes in the triclinic space group P&onemacr;, with a = 12.657(3) Å, b = 14.833(3) Å, c = 17.707(3) Å, alpha = 79.65(2) degrees, beta = 86.06(2) degrees, gamma = 68.69(2) degrees, V = 3046.6(12) Å, and Z = 2 (R1 = 0.0534, wR2 = 0.1289). Complex 2 crystallizes in the monoclinic space group P2(1)/n, with a = 16.319(2) Å, b = 21.863(2) Å, c = 18.410(3) Å, beta = 96.85(1) degrees, V = 6522(2) Å(3), and Z = 4 (R1 = 0.0463, wR2 = 0.0750). Complex 3 crystallizes in the triclinic space group P&onemacr;, with a = 11. 173(6) Å, b = 11.534(6) Å, c = 13.3...

Friedreich's ataxia, an autosomal cardio-and neurodegenerative disorder that affects 1 in 50,000 humans, is caused by decreased levels of the protein frataxin. Although nuclear encoded, frataxin is targeted to the mitochondrial matrix and necessary for proper regulation of cellular iron homeostasis. Frataxin is required for the cellular production of both heme and iron-sulfur clusters. Monomeric frataxin binds with high affinity to ferrochelatase, the enzyme involved in iron insertion into porphyrin during heme production. Monomeric frataxin also binds to Isu, the scaffold protein required for assembly of Fe-S cluster intermediates. These processes (heme and Fe-S cluster assembly) share requirements for iron, suggesting monomeric frataxin might function as the common iron donor.In order to provide a molecular basis to better understand frataxin's function, we have characterized the binding properties and metal site structure of ferrous iron bound to monomeric yeast frataxin. Yeast frataxin is stable as an iron loaded monomer and the protein can bind 2 ferrous iron atoms with micromolar binding affinity. Frataxin amino acids affected by the presence of iron are localized within conserved acidic patches located on the surfaces of both helix-1 and strand-1. Under anaerobic conditions, bound metal is stable in the high-spin ferrous state. The metal-ligand coordination geometry of both metal binding sites is consistent with a 6 coordinate iron-(oxygen and nitrogen) based ligand geometry, surely constructed in part from carboxylate and possibly imidazole side chains coming from residues within these conserved acidic patches on the protein. Based on our results, we have developed a model for how we believe yeast frataxin interacts with iron. Keywords Briefs:We present a characterization of monomeric yeast frataxin's iron binding ability. Various spectroscopic techniques were applied to help characterize the iron binding affinity of yeast frataxin, the oligomeric state of the protein, specific amino acids affected by the presence of iron and finally the metal-ligand coordination geometry. NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2008 August 19. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript patients, is a direct result of a trinucleotide repeat expansion in the first intron of the gene for the protein frataxin; this expansion disrupts gene transcription leading to a frataxin deficiency in humans (4). The inability to produce frataxin is associated with mitochondrial accumulation of iron that is biologically unavailable. In humans, the breakdown in normal iron regulation pathways resulting from frataxin deficiency is exemplified physiologically by the degeneration of sensory neurons in the dorsal root ganglia, hypertrophic cardiomyopathy and diabetes mellitus (5). In yeast, reintroduction of frataxin into knockout cells results in restored bioavailability of the accumulated metal (6). These data indicate frataxin is required for retaining bi...

No abstract

␤-Lactamases and penicillin-binding proteins are bacterial enzymes involved in antibiotic resistance to ␤-lactam antibiotics and biosynthetic assembly of cell wall, respectively. Members of these large families of enzymes all experience acylation by their respective substrates at an active site serine as the first step in their catalytic activities. A Ser-X-X-Lys sequence motif is seen in all these proteins, and crystal structures demonstrate that the side-chain functions of the serine and lysine are in contact with one another. Three independent methods were used in this report to address the question of the protonation state of this important lysine (Lys-73) in the TEM-1 ␤-lactamase from Escherichia coli. These techniques included perturbation of the pK a of Lys-73 by the study of the ␥-thialysine-73 variant and the attendant kinetic analyses, investigation of the protonation state by titration of specifically labeled proteins by nuclear magnetic resonance, and by computational treatment using the thermodynamic integration method. All three methods indicated that the pK a of Lys-73 of this enzyme is attenuated to 8.0 -8.5. It is argued herein that the unique ground-state ion pair of Glu-166 and Lys-73 of class A ␤-lactamases has actually raised the pK a of the active site lysine to 8.0 -8.5 from that of the parental penicillin-binding protein. Whereas we cannot rule out that Glu-166 might activate the active site water, which in turn promotes Ser-70 for the acylation event, such as proposed earlier, we would like to propose as a plausible alternative for the acylation step the possibility that the ion pair would reconfigure to the protonated Glu-166 and unprotonated Lys-73. As such, unprotonated Lys-73 could promote serine for acylation, a process that should be shared among all active-site serine ␤-lactamases and penicillin-binding proteins.A number of enzymes have evolved a catalytic strategy that depends on a transient acylation of an active site serine. The catalytic serine residue in these enzymes is followed by a lysine three residues toward the carboxyl termini of the proteins (i.e. . . . Ser-X-X-Lys . . . ). This sequence motif is seen in serinedependent ␤-lactamases and penicillin-binding proteins (PBPs 1 ), of which several hundred members are known. The catalytic implication of this Ser-X-X-Lys sequence motif for ␤-lactamases is debated in the literature, but the role of these residues in catalysis is likely to be general for the large group of proteins that share this sequence.␤-Lactamases are bacterial resistance enzymes to ␤-lactam antibiotics, which include penicillins and cephalosporins. Members of the class A ␤-lactamases are the most common among pathogenic bacteria. These enzymes undergo acylation and deacylation at Ser-70 during substrate turnover (1, 2). The process of deacylation of the acyl-enzyme intermediate is best understood. Glu-166 is the active-site general base that promotes a water molecule in the deacylation step (3-5). On the other hand, how the active-site serine experiences ...

ZntA from Escherichia coli, a P1-type ATPase, specifically transports Pb(II), Zn(II), and Cd(II). Most P1-type ATPases have an N-terminal domain that contains one or more copies of the conserved metal-binding motif, GXXCXXC. In ZntA, the N-terminal domain has approximately 120 residues with a single GXXCXXC motif, as well as four additional cysteine residues as part of the CCCDGAC motif. The metal-binding specificity and affinity of this domain in ZntA was investigated. Isolated proteins, N1-ZntA and N2-ZntA, containing residues 1-111 and 47-111 of ZntA, respectively, were characterized. N1-ZntA has both the CCCDGAC and GXXCXXC motifs, while N2-ZntA has only the GXXCXXC motif. ICP-MS measurements showed that N1-ZntA can bind both divalent metal ions such as Cd(II), Pb(II), and Zn(II) and monovalent metal ions such as Ag(I), with a stoichiometry of 1. N2-ZntA can bind Zn(II) and Cd(II) with a stoichiometry of 1 but not Pb(II). The affinity of N1-ZntA for Zn(II), Pb(II), and Cd(II) was measured by competition titration with metallochromic indicators. Association constants of approximately 10(8) M(-)(1) were obtained for Zn(II), Pb(II), and Cd(II) binding to N1-ZntA. To investigate whether the CCCDGAC sequence has an important role in binding specifically Pb(II), a mutant of ZntA, which lacked the first 46 residues, was constructed. This mutant, Delta46-ZntA, had the same activity as wtZntA with respect to Cd(II) and Zn(II). However, its activity with Pb(II) was similar to the mutant DeltaN-ZntA, which lacks the entire N-terminal domain (Mitra, B., and Sharma, R. (2001) Biochemistry 40, 7694-7699). Thus, binding of Pb(II) appears to involve different ligands, and possibly geometry, compared to Cd(II) and Zn(II).

No abstract

No abstract

The present results support the hypothesis that LPA can correct the impaired apparent ion demand in experimental diabetic retinopathy.