Redox Metal Ions Research Articles

Calcineurin: Form and Function

Calcineurin is a eukaryotic Ca(2+)- and calmodulin-dependent serine/threonine protein phosphatase. It is a heterodimeric protein consisting of a catalytic subunit calcineurin A, which contains an active site dinuclear metal center, and a tightly associated, myristoylated, Ca(2+)-binding subunit, calcineurin B. The primary sequence of both subunits and heterodimeric quaternary structure is highly conserved from yeast to mammals. As a serine/threonine protein phosphatase, calcineurin participates in a number of cellular processes and Ca(2+)-dependent signal transduction pathways. Calcineurin is potently inhibited by immunosuppressant drugs, cyclosporin A and FK506, in the presence of their respective cytoplasmic immunophilin proteins, cyclophilin and FK506-binding protein. Many studies have used these immunosuppressant drugs and/or modern genetic techniques to disrupt calcineurin in model organisms such as yeast, filamentous fungi, plants, vertebrates, and mammals to explore its biological function. Recent advances regarding calcineurin structure include the determination of its three-dimensional structure. In addition, biochemical and spectroscopic studies are beginning to unravel aspects of the mechanism of phosphate ester hydrolysis including the importance of the dinuclear metal ion cofactor and metal ion redox chemistry, studies which may lead to new calcineurin inhibitors. This review provides a comprehensive examination of the biological roles of calcineurin and reviews aspects related to its structure and catalytic mechanism.

Solution Structure of Copper Ion-Induced Molecular Aggregates of Tyrosine Melanin

Melanin, the ubiquitous biological pigment, provides photoprotection by efficient filtration of light and also by its antioxidant behavior. In solutions of synthetic melanin, both optical and antioxidant behavior are affected by the aggregation states of melanin. We have utilized small-angle x-ray and neutron scattering to determine the molecular dimensions of synthetic tyrosine melanin in its unaggregated state in D 2O and H 2O to study the structure of melanin aggregates formed in the presence of copper ions at various copper-to-melanin molar ratios. In the absence of copper ions, or at low copper ion concentrations, tyrosine melanin is present in solution as a sheet-like particle with a mean thickness of 12.5 Å and a lateral extent of ∼54 Å. At a copper-to-melanin molar ratio of 0.6, melanin aggregates to form long, rod-like structures with a radius of 32 Å. At a higher copper ion concentration, with a copper-to-melanin ratio of 1.0, these rod-like structures further aggregate, forming sheet-like structures with a mean thickness of 51 Å. A change in the charge of the ionizable groups induced by the addition of copper ions is proposed to account for part of the aggregation. The data also support a model for the copper-induced aggregation of melanin driven by π stacking assisted by peripheral Cu 2+ complexation. The relationship between our results and a previous hypothesis for reduced cellular damage from bound-to-melanin redox metal ions is also discussed.

Redox-modulated solute retention on iron–protoporphyrin–silica stationary phases

Iron–protoporphyrin (FeProP)–silica phases suitable for immobilized metal ion affinity chromatography (IMAC) are examined with respect to the change in solute retention behavior induced by switching the redox state of the central metal ion from Fe(III) to Fe(II). UV–Vis spectroscopy of a stationary phase slurry in typical mobile phases is employed to probe the conditions required for efficient chemical reduction and re-oxidation of the immobilized FeProP. Reduction of a column packed with sterically protected Fe(III)ProP–silica via dithionite and re-oxidation of the immobilized ferroheme with ferricyanide enables tuning of the retention behavior for a range of test solutes under reversed-phase conditions. Upon reduction, a significant effect on the retention of solutes that can coordinate to the central metal ion occurs, as evidenced by the behavior of imidazole, with k′ decreasing more than 50% following reduction. However, non-coordinating solutes (e.g., tryptophan, phenylalanine) also exhibit altered retention on Fe(II)–ProP stationary phases, demonstrating that there are, in addition, secondary effects of the metal ion redox state, most likely on the strength of π–π interactions of solutes with the immobilized porphyrin species.

Isolation, cloning, sequence analysis and X-ray structure of dimethyl sulfoxide/trimethylamine N-oxide reductase from Rhodobacter capsulatus.

The periplasmic enzyme dimethyl sulfoxide/trimethylamine N-oxide reductase (DMSOR/TMAOR) from the photosynthetic purple bacterium Rhodobacter capsulatus functions as the terminal electron acceptor in its respiratory chain. The enzyme catalyzes the reduction of highly oxidized substrates like dimethyl sulfoxide (DMSO) or trimethylamine N-oxide (TMAO). At a molybdenum redox centre, two single electrons are transferred from cytochrome c556 to the substrate, e.g. DMSO, generating dimethyl sulfide (DMS) and water. The operon encoding this enzyme was isolated, cloned and sequenced, and its chromosomal location determined. It was shown by analytical and crystallographic data that DMSOR and TMAOR are identical enzymes. Degenerate primers were derived from short peptide sequences and a 700 bp fragment was amplified by nested PCR, subsequently cloned and radioactively labeled to screen a prepared lambda DASH library. Positive lambda clones were subcloned into pBluescript and subsequently transformed into Escherichia coli to sequence the DMSOR/TMAOR operon. By an optimized protein purification high yields (5 mg protein/l culture) with a specific activity of 30 U/mg were obtained. The molecular mass was experimentally determined by electrospray mass spectroscopy (MS) to be 85034 Da and from the deduced amino acid sequence of the apoenzyme to be 85033 Da. The enzyme was crystallized in space group P4(1)2(1)2 with unit cell dimensions of a = b = 80.7 A and c = 229.2 A diffracting beyond 1.8 A. The three-dimensional structure was solved by a combination of multiple isomorphous replacement (MIR) and molecular replacement techniques. The atomic model was refined to an R-factor of 0.169 for 57394 independent reflections. The spherical protein consists of four domains with a funnel-like cavity that leads to the freely accessible metal-ion redox center. The sole bis(molybdopterin guanine dinucleotide)molybdenum cofactor (1541 Da) of the single chain protein has the molybdenum ion bound to the cis-dithiolene group of only one molybdopterin guanine dinucleotide (MGD) molecule. In addition, two oxo ligands and the oxygen of a serine side chain are bound to the molybdenum ion.

COMPACT and molecular structure in toxicity assessment: a prospective evaluation of 30 chemicals currently being tested for rodent carcinogenicity by the NCI/NTP.

A new series of 30 miscellaneous National Toxicology Program chemicals has been evaluated prospectively for carcinogenicity and overt toxicity by COMPACT (Computer Optimised Molecular Parametric Analysis for Chemical Toxicity. CYP1A and CYP2E1). Evaluations were also made by Hazardexpert, and for metal ion redox potentials; and these, together with COMPACT, were compared with results from the Ames test for mutagenicity in Salmonella, the micronucleus test, and 90-day subchronic rodent pathology. Seven of the 30 chemicals (nitromethane, chloroprene, xylenesulphonic acid, furfuryl alcohol, anthraquinone, emodin, cinnamaldehyde) were positive for potential carcinogenicity in the COMPACT evaluation; xylenesulphonic acid and furfuryl alcohol were only equivocally positive. Four of the 30 chemicals-scopolamine, D&C Yellow No. 11, citral, cinnamaldehyde-were positive by Hazardexpert; 6 of 30-D&C Yellow No. 11, 1-chloro-2-propanol, anthraquinone, emodin, sodium nitrite, cinnamaldehyde-were positive in the Ames test; 2 of 30-phenolphthalein and emodin-were positive in the in vivo cytogenetics test; and 3 of 30-molybdenum trioxide, gallium arsenide, vanadium pentoxide-were metal compounds with redox potentials of the metal/metal ion indicative of possible carcinogenicity. The overall prediction for carcinogenicity was positive for 12 of 30 chemicals: nitromethane, chloroprene, D&C Yellow No. 11, molybdenum trioxide, 1-chloro-2-propanol, furfuryl alcohol, gallium arsenide, anthraquinone, emodin, sodium nitrite, cinnamaldehyde, vanadium pentoxide). This overall prediction has been made on the basis of the results of the computer tests and from consideration of the information from bacterial mutagenicity, together with likely lipid solubility and pathways of metabolism and elimination.

Crystal Structure of Dimethyl Sulfoxide Reductase from Rhodobacter capsulatusat 1.88 Å Resolution

The periplasmic dimethyl sulfoxide reductase (DMSOR) from the photosynthetic purple bacterium Rhodobacter capsulatusfunctions as the terminal electron acceptor in its respiratory chain. The enzyme catalyzes the reduction of highly oxidized substrates like dimethyl sulfoxide to dimethyl sulfide. At a molybdenum redox center, two single electrons are transferred from cytochrome c 556to the substrate dimethyl sulfoxide, generating dimethyl sulfide and (with two protons) water. The enzyme was purified and crystallized in space group P4 12 12 with unit cell dimensions of a= b=80.7 Å and c=229.2 Å. The crystals diffract beyond 1.8 Å with synchrotron radiation. The three-dimensional structure was solved by a combination of multiple isomorphous replacement and molecular replacement techniques. The atomic model was refined to an R-factor of 0.169 for 57,394 independent reflections. The spherical protein consists of four domains with a funnel-like cavity that leads to the freely accessible metal-ion redox center. The bis(molybdopterin guanine dinucleotide)molybdenum cofactor (1541 Da) of the single chain protein (85,033 Da) has the molybdenum ion bound to the cis-dithiolene group of only one molybdopterin guanine dinucleotide molecule. Three additional ligands, two oxo groups and the oxygen of a serine side-chain, are bound to the molybdenum ion. The second molybdopterin system is not part of the ligand sphere of the metal center with its sulfur atoms at distances of 3.5 Å and 3.8 Å away. It might be involved in electron shuttling from the protein surface to the molybdenum center.

Effect of aluminum on the non-enzymatic oxidation of dopamine

The effect of different concentrations of aluminum sulphate on the nonenzymatic oxidation of dopamine was studied in order to evaluate the action of this metal on neuromelanin synthesis. Data shows that under the studied conditions, aluminum partially inhibits dopamine self-oxidation, decreasing the formation of some intermediate compounds, namely dopaminequinone and dopaminochrome. If neuromelanins have a cytoprotective function in the central nervous system, possibly acting as intracellular scavengers of free radicals and redox metal ions, their decrease due to aluminum could be responsible for serious damage to neuronal tissues.

A Consideration of the Indirect Electrooxidation of Alkylbenzenes by Metal-Ion Mediators

Abstract Selective oxidation of substituted alkylbenzenes to the corresponding aromatic aldehydes by metal-ion redox mediators has been investigated. The apparent oxidation potential of the substrate alkylbenzene (Es,ox) measured by voltammetry in an organic medium was used as a measure of the substrate reactivity. The oxidizing power of the mediator was also evaluated based on the apparent reduction potential in the voltammogram (Ered). The yields of the experimentally determined reaction products were explained by the mutual relation between Es,ox of the substrate and Ered of the mediator.

Stoichiometry and redox behaviour of metals in cytochrome-c oxidase.

The early observation of extra copper in preparations of cytochrome-c oxidase has recently lead to a renewed interest in its stoichiometry and possible redox function. In various, pure preparations (heme A contents close to the theoretical value of 9.79 nmol/mg protein for the 13-subunit bovine enzyme) protein-related metal stoichiometries of 3 Cu, 2 Fe, 1 Zn, 1 Mg/monomer with M(r) 204266 were determined. Despite the presence of five potential redox metal ions, reductive and reoxidative titrations indicate the presence of only four one-electron-accepting/donating species in the ligand-free enzyme. Participation of two copper ions in a binuclear copper site acting as one-electron acceptor may explain both the observed copper stoichiometry and the redox behaviour. The homology of the C-terminal sequence of subunit II with one of the copper-binding sites in nitrous-oxide reductases provides possible ligands for complexing two copper ions in a binuclear center.

Developments in functionalization of macrocyclic polyamines

Abstract

Kinetics and mechanism of hydrolysis of acetamides in metal-ion redox systems

The hydrolysis ofN-methylacetamide andN,N-dimenthylacetamide in the presence of cobalt(III) and cerium(IV) has been studied in perchloric acid in the range 10–50° C. The reduction of metal ions and the hydrolysis of amides are very slow processes. However, it appears that reduction of metal ion is coupled with the hydrolysis of amides in such a way as to give enhanced rates for the two processes. The reaction requires one mole of the metal ion for one mole of amide. The rate is proportional to the acid concentration and is independent of ionic strength. Activation parameters have been calculated and reaction mechanisms proposed.

Vinyl polymerization with metal ion redox systems as initiators, manganic laurate—thiol system

Polymerization of methyl methacrylate was studied using manganic laurate in the presence of organic thiols as initiators. The thiols used were 1-propane thiol and 2-propane thiol and the solvent was carbon tetrachloride. The rate of polymerization satisfied the expression R P = k 1 [manganic laurate] 1/2 [thiol] 1/2 [monomer] 3/2 where k 1 is a constant. Spectroscopic investigations indicated complex formation between the monomer and manganic laurate. A reaction scheme is suggested.

Mechanisms of lipid peroxidation

This article provides an overview of how peroxidation of unsaturated lipids takes place and how it can be measured. Several different aspects of free-radical-mediated lipid peroxidation are discussed, including: (a) the catalytic role of chelated iron and other redox metal ions; (b) induction by reducing agents such as superoxide, ascorbate, and xenobiotic free radicals; (c) suppression by antioxidant chemicals and enzymes; and (d) how peroxidation that depends on pre-existing hydroperoxides (lipid hydroperoxide-dependent initiation of lipid peroxidation) can be distinguished from that which does not (lipid hydroperoxide-independent initiation of lipid peroxidation). Attention is also given to non-radical, singlet oxygen-driven peroxidation and how this can be resolved from radical-driven processes.

Cyanide formation from histidine in Chlorella a general reaction of aromatic amino acids catalyzed by amino acid oxidase systems

The formation of HCN from d-histidine in Chlorella vulgaris extracts is shown to be due to the combined action of a soluble protein and a particulate component. Either horse-radish peroxidase (EC 1.11.1.7) or a metal ion with redox properties can be substituted for the particulate component. Ions of manganese and vanadium are especially effective, as are o- phenanthroline complexes of iron. Cobalt ions are less active. The d-amino acid oxidase (EC 1.4.3.3) from kidney and the l-amino acid oxidase (EC 1.4.3.2) from snake venom likewise cause HCN production from histidine when supplemented with the particulate preparation from Chlorella or with peroxidase or with a redox metal ion. The stereospecificity of the amino acid oxidase determines which of the two stereoisomers of histidine is active as an HCN precursor. Though histidine is the best substrate for HCN production, other naturally occurring aromatic amino acids (viz. tyrosine, phenylalanine and tryptophan) can also serve as HCN precursors with these enzyme systems. The relative effectiveness of each substrate varies with the amino acid oxidase enzyme and with the supplement. With respect to this latter property, the particulate preparation from Chlorella behaves more like a metal ion than like peroxidase.

Metal ion redox systems as radiation protective agents.

IN investigations of the sensitivity of enzymes to ionizing radiations both in the solid state and in solution a marked variability has been observed between different batches of one enzyme1. This has been ascribed to the presence of protective agents which are impurities even in samples of high enzymatic activity, and which are often difficult to remove. Little has been done to identify such natural agents although various substances, particularly sulphydryl compounds, have long been recognized as being especially effective as protective agents. It was suggested by Howard-Flanders2 that these compounds act by donating hydrogen atoms to the radicals formed under the action of radiation, thus repairing the initial damage.