What is a metal ion ?

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What is a metal ion ?

Metal Ions in Biochemistry presents a simplified account of the role of metal ions in the biochemical processes and the significance of inorganic elements in human diet and in therapeutics.

Anintroductiontofreeradicalbiochemistry

Free radicals are chemical species possessing an unpaired electron that can be considered as fragments of molecules and which are generally very reactive. They are produced continuously in cells either as accidental by-products of metabolism or deliberately during, for example, phagocytosis. The most important reactants in free radical biochemistry in aerobic cells are oxygen and its radical derivatives (superoxide and hydroxyl radical), hydrogen peroxide and transition metals. Cells have developed a comprehensive array of antioxidant defences to prevent free radical formation or limit their damaging effects. These include enzymes to decompose peroxides, proteins to sequester transition metals and a range of compounds to ‘scavenge' free radicals. Reactive free radicals formed within cells can oxidise biomolecules and lead to cell death and tissue injury. Establishing the involvement of free radicals in the pathogenesis of a disease is extremely difficult due to the short lifetimes of these species.

Metal ions play a passive role in the hairpin ribozyme catalysed reaction.

The hairpin ribozyme is an example of a small catalytic RNA which catalyses the endonucleolytic transesterification of RNA in a highly sequence-specific manner. The hairpin ribozyme, in common with all other small ribozymes such as the hammerhead, requires the presence of a divalent metal ion co-factor (typically magnesium) for the reaction to take place. To investigate the role of magnesium ions in the hairpin catalysed reaction we have synthesised two epimeric modified substrates in which a phosphorothioate replaces the scissile phosphodiester bond. Previously, Burke and co-workers have reported that no thio-effect is observed with the Rp-phosphorothioate isomer. We observe the absence of a thio-effect with both diastereomeric phosphorothioate hairpin substrates. Furthermore we report that inert cobalt (III) complexes are capable of supporting the hairpin ribozyme reaction, with a similar efficiency to Mg2+,even in the presence of EDTA. Variation of the net charge on the inert cobalt complex does not change the observed rate of reaction. These results suggest that metal ions play a passive role in the hairpin ribozyme catalysed reaction and are probably required for structural purposes only. This places the hairpin ribozyme in a different mechanistic class to other small ribozymes such as the hammerhead.

Role of the divalent metal ion in the NAD:malic enzyme reaction: an ESEEM determination of the ground state conformation of malate in the E:Mn:malate complex.

Conformation of L-malate bound at the active site of Ascaris suum malic enzyme has been investigated by electron spin echo envelope modulation spectroscopy. Dipolar interactions between Mn2+ bound to the enzyme active site and deuterium specifically placed at the 2-position, the 3R-position, and the 3S-position of L-malate were observed. The intensities of these interactions are related to the distance between each deuterium and Mn2+. Several models of possible Mn-malate complexes were constructed using molecular graphics techniques, and conformational searches were conducted to identify conformers of malate that meet the distance criteria defined by the spectroscopic measurements. These searches suggest that L-malate binds to the enzyme active site in the trans conformation, which would be expected to be the most stable conformer in solution, not in the gauche conformer, which would be more similar to the conformation required for oxidative decarboxylation of oxalacetate formed from L-malate at the active site of the enzyme.Effect of A and B Metal Ion Site Occupancy on Conformational Changes in an RB69 DNA Polymerase Ternary Complex

Rapid chemical quench assays, as well as equilibrium and stopped-flow fluorescence experiments, were performed with an RB69 DNA polymerase (RB69 pol)−primer-template (P/T) complex containing 2-aminopurine (dAP) and a metal exchange-inert Rh(III) derivative of a deoxynucleoside triphosphate (Rh·dTTP). The objective was to determine the effect of catalytic metal ion (A site) occupancy on the affinity of an incoming Rh·dTTP for the RB69 pol−P/T binary complex and on the rate of the conformational change induced by Rh·dTTP binding. With Ca2+ in the A site, the affinity of the incoming Rh·dTTP for the RB69 pol−P/T binary complex and the conformational change rate can be determined in the absence of chemistry. When Mg2+ was added to a ternary complex containing Rh·dTTP opposite dAP, the templating base, nucleotidyl transfer occurred, but the rate of product formation was only one-tenth of that found with Mg·dTTP, as determined by rapid chemical quench assays. Rates of conformational change subsequent to formation of a ternary complex, in the absence of chemistry, were estimated from the rate of change in dAP fluorescence with an increase in the Rh·dTTP concentration. We have shown that there is an initial rapid quenching of dAP fluorescence followed by a second phase of dAP quenching, which has nearly the same rate as that of dTMP incorporation, as estimated from rapid chemical quench experiments. We have also demonstrated that the affinity of Rh·dTTP for occupancy of the B metal ion site is dependent on the presence of Ca2+. However, a saturating Rh·dTTP concentration in the absence of Ca2+ results in full quenching of dAP fluorescence, whereas a saturating Ca2+ concentration in the absence of Rh·dTTP gives only partial quenching of dAP fluorescence. The implications of these results for the mechanism of Fingers closing, metal ion binding, and base selectivity are discussed.

Effect of Metal Ion on theStructural Stability of Tumour Suppressor Protein p53 DNA-Binding Domain

The tumour suppressor protein p53 is a sequence-specific transcription factor that coordinates one molecule of zinc in the core domain. In our recent study, magnesium can also bind to the p53DBD and enhance its DNA-binding activity. In this study, a systematic analysis of the conformation and stability changes induced by these two metal ions was reported. The spectra of protein intrinsic fluorescence were used to measure the equilibrium unfolding of the p53DBD protein. The stability against chemical denaturation increased in the order apo < Mg2+ < Zn2+. The thermal stability monitored by DSC scans showed that the binding of metal ions to p53DBD increased the thermal stability of the protein. To explore additional information of structural changes after the binding of metal ions, we used the fluorescent probes to evaluate the hydrophobic surface exposure. The results established that metal ions binding increased hydrophobic exposure on the surface of p53DBD. Analysis of acrylamide quenching experiments revealed that the binding of metal ions to p53DBD induced a structural modification of the protein and this change provided significant protection against acrylamide quenching. Overall, the present results indicated that p53DBD underwent a conformational change upon the binding of metal ions, which was characterized by an increased stability of the protein.

Abbreviations: ANS, 1-anilino-8-naphthalene sulphonate; DBD, DNA-binding domain; DSC, differential scanning calorimetry; DTT, dithiothreitol; GdmHCl, guanidinium hydrochloride; SDS, sodium dodecyl sulphate

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