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Specific interactions of lipoate at the active site of rhodanese
No full textDihydrolipoate is an acceptor of the rhodanese-bound sulfane sulfur atom, as shown by analysis of the elementary steps of the reaction catalyzed by rhodanese. The crystal structure of sulfur-substituted rhodanese complexed with the non-reactive oxidized form of lipoate has revealed that the compound is bound at the enzyme active site, with the dithiolane ring buried in the interior of the cavity and the carboxylic end pointing towards the solvent. One of the sulfur atoms of the ligand in the unproductive complex is relatively close to the sulfane sulfur bound to Cys-247, the sulfur that is transferred during the catalytic reaction. This mode of binding of lipoate is likely to mimic that of dihydrolipoate. The results presented here support the possible role of dihydrolipoate as sulfur-acceptor substrate of rhodanese in an enzymatic reaction that might serve to provide iron-sulfur proteins with inorganic sulfide
Structure of sulfur-substituted rhodanese at 1.36 Angstrom resolution
No full text1.36 Angstrom resolution X-ray diffraction data have been recorded at 100 K for bovine liver sulfur-substituted rhodanese, using synchrotron radiation. The crystal structure has been refined anisotropically to a final R factor of 0.159 (R-free = 0.229) for 53 034 unique reflections. The model contains 2 327 protein atoms and 407 solvent molecules, with a good geometry. The high resolution allows full details for helices, beta-sheets, tight turns and of all inter- and intramolecular interactions stabilizing the enzyme molecule to be given. The situation at the active site is described, particularly in regard to the network of hydrogen bonds made by S-gamma and S-delta of the sulfur-substituted catalytic Cys247 and surrounding groups and solvent molecules. The replacement of the precipitant ammonium sulfate with cryoprotectants in the crystal-suspending medium led to the removal of the sulfate ion from the enzyme active site. Only limited changes of the enzyme structure have been found as a result of the drastic change in the crystal medium
NH2-terminal sequence truncation decreases the stability of bovine rhodanese, minimally perturbs its crystal structure and enhances interaction with groel under native conditions
No full textActive site structural features for chemically modified forms of rhodanese
No full textIn the course of the reaction catalyzed by rhodanese, the enzyme cycles between two catalytic intermediates, the sulfur-free and the sulfur-substituted (persulfide-containing) forms. The crystal structure of sulfur-free rhodanese, which was prepared in solution and then crystallized, is highly similar to that of sulfur-substituted enzyme. The inactivation of sulfur-free rhodanese with a small molar excess of hydrogen peroxide relies essentially on a modification limited to the active site, consisting of the oxidation of the essential sulfhydryl to sulfenyl group (-S-OH). Upon reaction of the sulfur-free enzyme with monoiodoacetate in the crystal, the Cys-247 side chain with the bound carboxymethyl group is forced into a conformation that allows favorable interactions of the carboxylate with the four peptide NH groups that participate in hydrogen bonding interactions with the transferable sulfur atom of the persulfide group in the sulfur-substituted rhodanese. It is concluded that active site-specific chemical modifications of sulfur-free rhodanese do not lead to significant changes of the protein structure, consistent with a high degree of similarity of the structures of the sulfur-free and sulfur-substituted forms of the enzyme both in solution and iu the crystal
Crystal Structure of Bovine 3-Hydroxyanthranilate 3,4-Dioxygenase
No full text3-Hydroxyanthranilate 3,4-dioxygenase, the enzyme that catalyzes the conversion of 3-hydroxyanthranilate to quinolinic acid, has been extracted and purified from bovine kidney, crystallized and its structure determined at 2.5 Å resolution. The enzyme, which crystallizes in the triclinic P1 space group, is a monomer, characterized by the so-called cupin fold. The monomer of the bovine enzyme mimics the dimer present in lower species, such as bacteria and yeast, since it is composed of two domains: one of them is equivalent to one monomer, whilst the second domain corresponds to only a portion of it. The active site consists of an iron ion coordinated by two histidine residues, one glutamate and an external ligand, which has been interpreted as a solvent molecule. It is contained in the N-terminal domain, whilst the function of the C-terminal domain is possibly structural. The catalytic mechanism very likely has been conserved through all species, since the positions of all residues considered relevant for the reaction are present from bacteria to humans
Cytochrome c Oxidase Models - Synthesis and Reactivity of Iron(III)/Copper(II) Complexes of Deuterohaemin-Polybenzimidazole Dinucleating Ligands
No full textDeuterohaemin complexes modified through covalent linkage of bis- (1 ) or tris-(benzimidazole) (2)
residues to one of the propionic acid side chains have been obtained and spectrally characterised. While in
1 the benzimidazole groups cannot bind intramolecularly to the iron centre, in 2 such an arrangement
is possible because the carbon chain connecting the aromatic donor group to the propionic carbonyl
group is slightly longer. As a consequence, while complex 1 binds two molecules of exogenous
donor bases in two steps for 2 simultaneous binding affinity for two donor bases is very low. The
intramolecular binding of benzimidazole markedly increases the reactivity of 2 in catalytic peroxidative
reactions. Both complexes bond one copper(1i) ion at the polybenzimidazole site. For 2 some
competition between iron(iii) and copper(ii) for one of the ligand donor bases is evident from EPR
measurements. Reduction experiments show that upon treatment with sodium ascorbate the partially
reduced Fe"'-Cul complex is obtained, while the stronger reductant sodium dithionite produces the
fully reduced FeIl-Cu' complex. Oxidation of the latter with dioxygen at low temperature (about
-40 "C) occurs without any destruction of the porphyrin and gives the fully oxidised Fe"'-Cu" complex.
The EPR spectra (-1 50 "C) of the ironxopper complexes show a decrease in intensity of the Cu"
signal upon addition of small amounts of OH-, but the marked tendency towards intermolecular
association prevents a simple interpretation in terms of formation of an intramolecular Fe"'-OH--Cu"
bridg
Crystallization and preliminary X-ray data for the human transthyretin-retinol-binding protein complex bound to and anti-RBP Fab
No full textCrystallization and preliminary X-ray data for the human transthyretin-retinol-binding protein (RBP) complex bound to an anti-RBP FAB
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