Complexes of hydroxamates. Part 1. Interaction of methioninehydroxamic acid with iron(III) in aqueous solution

The binding affinity of Fe(III) to methioninehydroxamate (MX) has been studied spectrophotometrically at I=0.15 M NaCl and T=25 °C. Equilibrium data have been assessed by the program SQUAD(II) in the wavelength range 400–550 nm and in the pH range 1.5–5.0. Five formation constants were determined for the species Fe(MX)(H)³⁺, Fe(MX)²⁺, Fe(MX)₂(H)₂³⁺, Fe(MX)₂(H)²⁺ and Fe₂(MX)₃³⁺. The stopped-flow kinetic data studied at 470 nm and in the pH range 1.0–3.0 is collectively expressed by the following rate equation at a given pH Rate=(A + BT_MX)T_MX where T_MX=the analytical connection of MX and the parameters A and B are both functions of pH in the range 1.7–3.0, but only A in the range 1.2– 1.7. A proposed mechanism was discussed, based on the equilibrium study, where the role of the chloro species of Fe(OH)²⁺ and Fe(OH)₂⁺ in the complex formation of Fe(III) with MX has been emphasized. Correlation of the results with pertinent systems has also been discussed.     

The dimerization of ferrihaems: III. Equilibrium and kinetic studies on the dimerization of coproferrihaem

Spectrophotometric studies on the behaviour of coproferrihaem in aqueous solution showed that, in the pH range 6.66–8.04, a dimerization process occurs according to the equation 2 monomer

The value of K, the pH-independent dimerization contant, was found to be 2.10 · 10^?3, signifying that coproferrihaem shows the least tendency to dimerize of any ferrihaem so far investigated. Forward and reverse rate constants for the dimerization process have been determined by the temperature-jump method.The results suggest that the cation-briding between carboxyl residues, postulated for the dimers of the dicarboxylic ferrihaems, cannot occur between the additional carboxyl residues of coproferrihaem and that the increased negative charge may cause destabilization of the coproferrihaem dimer by repulsion effects.     

The dimerization of ferrihaems. III. Equilibrium and kinetic studies on the dimerization of coproferrihaem.

Spectrophotometric studies on the behaviour of coproferrihaem in aqueous solution showed that, in the pH range 6.66--8.04, a dimerization process occurs according to the equation 2 monomer K in equilibrium dimer + H+ The value of K, the pH-independent dimerization constant, was found to be 2.10 . 10(-3), signifying that coproferrihaem shows the least tendency to dimerize of any ferrihaem so far investigated. Forward and reverse rate constants for the dimerization process have been determined by the temperature-jump method. The results suggest that the cation-bridging between carboxyl residues, postulated for the dimers of the dicarboxylic ferrihaems, cannot occur between the additional carboxyl residues of coproferrihaem and that the increased negative charge may cause destabiliztion of the coproferrihaem dimer by repulsion effects.     

Kinetic studies on the interaction of gold (III) with nucleic acids. IV. RNA-Au (III) system.

The kinetics of the interaction of Au(III) with whole yeast RNA has been studied using UV-spectrophotometry. The reaction is second order with respect to the nucleotide unit of RNA and first order with respect to Au(III) in the respective stoichiometry of 2 : 1. The effects of initial composition, temperature, ionic strength, pH and chloride ion on the kinetics have been studied. Activation energy is found to be 11.5 kcal/mol. Effect of ionic strength indicates that both the positively charged and neutral species of Au(III) take part in the rate limiting step, the former being dominant at low ionic strength. A plausible mechanism has been proposed which involves the interaction of two nucleotide units of RNA with one species of Au(III) in the rate limiting step.     

The reactions of octaethylporphyrin and its iron (II) and iron (III) complexes with free radicals

The reactions of dilute solutions of octaethylporphyrin and its iron (II) and iron (III) complexes with methyl, 2-cyanopropyl, t-butoxy, and benzoyloxy radicals are described. The results are summarized: (i) The reactivity of the porphyrin and its high-spin iron (II) and iron (III) complexes toward alkyl and t-butoxy radicals stands in the order: Fe^II > Fe^III ? free porphyrin. For benzoyloxy radicals the order is Fe^II > Porp > Fe^III. (ii) The exclusive path of reaction of high-spin iron (II) porphyrin with radicals is the rapid reduction of the radical and generation of an iron (III) porphyrin. The dominant path of reaction of high-spin iron (III) porphyrin with alkyl and (presumably) t-butoxy radicals is a rapid axial inner sphere reduction of the porphyrin. An axial ligand of iron is transferred to the radical. (iv) The reaction of benzoyloxy radicals with high or low-spin iron (III) porphyrins occurs primarily at the meso position. With the low-spin dipyridyl complex in pyridine the attendant reduction to iron (II) can be observed spectrally. Methyl radicals also reduce this complex by adding to the meso position. (v) The reaction of a radical with either an iron (II) or an iron (III) porphyrin results in the generation of the other valence state of iron and consequently oxidation and reduction products emanating from both iron species are obtained. (vi) No evidence for an iron (IV) is intermediate is apparent. (vii) Iron (II) porphyrins in solvents that impart either spin state are easily oxidized by diacyl peroxides. The occurrence of both axial and peripheral redox reactions with the iron complexes supports an underlying premise of a recent theory of hemeprotein reactivity. The relevance of the work to bioelectron transfer and heme catabolism is noted.     

Complexes of dibenzoyldisulphide with metal halides. Part III. Complexes of dibenzoyldisulphide with cobalt(II), nickel(II), manganese(II), copper(II), iron(III) and chromium(III) halides

Adducts (1:1) of halides of cobalt(II), nickel(II), manganese(II), copper(II), iron(III) and chromium(III) with dibenzoyldisulphide have been isolated and characterized on the basis of elemental analysis, molar conductance, magnetic susceptibility, infrared spectra, molecular weight and thermogravimetric analysis data.     

Equilibrium and kinetic studies of ligand BMXD complexation with copper(II) and glycylglycine

Equilibrium studies of BMXD complexation with copper(II) and glycylglycine were performed by potentiometric and UV-visible titration. The equilibrium constants determined by potentiometry for the ternary system are: [LCu₂P³⁺]/[LCu₂⁴⁺][P⁻]=8.11(3), [LCu₂HP⁴⁺]/[LCu₂P³⁺][H⁺]=5.39(2), [LCu₂H₋₁P²⁺][H⁺]/[LCu₂P³⁺]=−6.33(2), [LCu₂(OH)H₋₁P⁺][H⁺]/[LCu₂H₋₁P²⁺]=−8.20(1) and [LCu₂(OH)₂HH₋₁P][H⁺]/[LCu₂(OH)H₋₁P⁻¹]=−10.33(3), which are in good agreement with the values determined by UV-visible titration. The species formed were confirmed by electrospray mass spectrometry. The association constants, determined by kinetic studies, for the association of the second molecule of glycylglycine with the LCu₂P complex, for two ionic media, are 1.29 (KCl) and 1.27 (KNO₃). These are in good agreement with the values determined by thermodynamic measurements 1.41 (KCl) and 1.26 (KNO₃).     

The kinetics and mechanisms of the reactions of iron(III) with quercetin and morin

The kinetics and mechanisms of the reactions of a pseudo-first order excess of iron(III) with the flavonoids quercetin and morin have been investigated in aqueous solution at 25 degrees C and an ionic strength of 0.5M. Mechanisms have been proposed which account satisfactorily for the kinetic data. The data are consistent with a mechanism in which the metal:ligand complex formed initially on reaction of iron(III) with the ligand subsequently decomposes through an electron transfer step. Morin forms a 1:1 metal:ligand complex while quercetin forms a 2:1 metal:ligand complex. Both ligands showed evidence for the involvement of the iron hydroxo dimer Fe2(OH)2(4+) in the complex formation reaction at the hydroxy-carbonyl moiety. The iron(III) assisted decomposition of the initial iron(III) complex formed was also investigated and the rate constants evaluated. Both the complex formation and subsequent electron transfer reactions of iron(III) with these ligands were monitored using UV-visible spectrophotometry. All of the suggested mechanisms and calculated rate constants are supported by calculations carried out using global analysis of time dependant spectra.     

Equilibrium and rapid kinetic studies on nocodazole-tubulin interaction

The interaction between nocodazole and calf brain tubulin in 10(-2) M sodium phosphate, 10(-4) M GTP, and 12% (v/v) dimethyl sulfoxide at pH 7.0 was studied. The number of binding sites for nocodazole was shown to be one per tubulin monomer of 50,000 as a result of equilibrium binding studies by gel filtration and spectroscopic techniques. The presence of microtubule-associated proteins did not significantly affect the binding of nocodazole to tubulin. The apparent equilibrium constant measured at 25 degrees C was (4 +/- 1) X 10(5) M-1. Temperature does not significantly affect the apparent equilibrium constant; hence, the binding of nocodazole to tubulin is apparently entropy driven. Stopped flow spectroscopy was employed to monitor the rate of nocodazole binding under pseudo first order conditions. The effects of temperature and nocodazole concentration were studied. The apparent rate constants were dependent on the concentration of nocodazole in a nonlinear manner. In conjunction with results from structural and thermodynamic studies the kinetic results were interpreted to suggest a mechanism of T + N in equilibrium with TN in equilibrium with T* N, where T and N are tubulin and nocodazole, respectively. T and T* represent two conformational states of tubulin. Furthermore, the kinetic data are consistent with the thermodynamic data only if a model of two parallel similar reactions were considered, one rapid and the other slow. The initial binding step for both the rapid and slow phases was characterized by identical binding constants; however, there was a significant difference in the rates of isomerization. Hence, nocodazole is potentially a useful probe for amplifying differences in solution properties of tubulin subspecies.     

Equilibrium and kinetic studies on the interaction of tetracyclines with calcium and magnesium

The interaction of Ca2+ and Mg2+ with three Tetracycline antibiotics (tetracycline, chlorotetracycline, and oxytetracycline) has been investigated. Spectrophotometric measurements have been used to determine the apparent association constant for this interaction as a function of pH. It is shown that the results are consistent with a model in which the metal ion can form complexes with both the fully-deprotonated and mono-protonated forms of the Tetracycline. The temperature-jump relaxation method has been used to measure the kinetics of formation of the complexes of Mg2+ with the Tetracyclines. The results are compared with those of previous studies of Mg2+ complex formation reactions and it is shown that the data is consistent with the normal dissociative model. A possible role for metal ion chelation in the mechanism of antibacterial action of the Tetracyclines is discussed.     

Investigation of the reactions of iron(III) halobisdithiocarbamates with halogens: a novel series of fluxional homobinuclear iron(III) complexes with two different coordination spheres around the magnetic centers

In addition to the variety of products formed during the reaction of iron(III) halobisdithiocarbamates with halogens, some novel fluxional homobinuclear iron(III) complexes with two different coordination spheres around the magnetic centers have also been synthesized and studied. The formation of these products depends on the nature of both the molecular halogen and the reagent complex, as well as on the reaction conditions. The new compounds have been characterized chemically and by means of spectroscopic methods and magnetic susceptibility measurements. The volatility characteristics and thermogravimetric analysis data for the complexes were also studied. Finally, a general mechanism accounting for the variety of products formed in the reactions of iron(III) halobisdithiocarbamates with molecular halogens is proposed.     

Interaction between iron(II) and hydroxamic acids: oxidation of iron(II) to iron(III) by desferrioxamine B under anaerobic conditions

Interaction between iron(II) and acetohydroxamic acid (Aha), alpha-alaninehydroxamic acid (alpha-Alaha), beta-alaninehydroxamic acid (beta-Alaha), hexanedioic acid bis(3-hydroxycarbamoyl-methyl)amide (Dha) or desferrioxamine B (DFB) under anaerobic conditions was studied by pH-metric and UV-Visible spectrophotometric methods. The stability constants of complexes formed with Aha, alpha-Alaha, beta-Alaha and Dha were calculated and turned out to be much lower than those of the corresponding iron(II) complexes. Stability constants of the iron(II)-hydroxamate complexes are compared with those of other divalent 3d-block metal ions and the Irving-Williams series of stabilities was found to be observed. Above pH 4, in the reactions between iron(II) and desferrioxamine B, the oxidation of the metal ion to iron(III) by the ligand was found. The overall reaction that resulted in the formation of the tris-hydroxamato complex [Fe(HDFB)]+ and monoamide derivative of DFB at pH 6 is: 2Fe2+ + 3H4DFB+ = 2[Fe(HDFB)]+ + H3DFB-monoamide+ + H2O + 4H+. Based on these results, the conclusion is that desferrioxamine B can uptake iron in iron(III) form under anaerobic conditions.     

Equilibrium and kinetic studies of unfolding of homologous cytochromes c.

Extensive investigations of the unfolding equilibria and kinetics of oxidized and reduced cytochromes c are reported. It is found that all cytochromes c have similar unfolding free energies (deltaGD = 7 +/- 1 kcal/mol). Differences among species do not correlate in any way with the metabolic differences among species. The stabilization of cytochrome c on reduction is estimated at 1.1 kcal/mol. Stability differences among species are mirrored in their denaturation kinetics. For cytochrome c (III), the unfolding exhibits multiple phases. The rate constants for the two observable phases both change by a factor of 3 between horse cytochrome c (III) and cow cytochrome c (III). On reduction, all unfolding appears to occur in a single step. The rate of this unfolding still varies between species, however, the results can be accommodated to a sequential model, with some assumptions. The observations are consistent with chain reversal occurring at an early stage in the reaction and suggest that previously observed rapid processes may be ligand exchange processes.     

Nature of the fast and slow refolding reactions of iron(III) cytochrome c

The fast and slow refolding reactions of iron(III) cytochrome c (Fe(III) cyt c), previously studied by Ikai et al. (Ikai, A., Fish, W. W., & Tanford, C. (1973) J. Mol. Biol. 73, 165--184), have been reinvestigated. The fast reaction has the major amplitude (78%) and is 100-fold faster than the slow reaction in these conditions (pH 7.2, 25 degrees C, 1.75 M guanidine hydrochloride). We show here that native cyt c is the product formed in the fast reaction as well as in the slow reaction. Two probes have been used to test for formation of native cyt c. absorbance in the 695-nm band and rate of reduction of by L-ascorbate. Different unfolded species (UF, US) give rise to the fast and slow refolding reactions, as shown both by refolding assays at different times after unfolding ("double-jump" experiments) and by the formation of native cyt c in each of the fast and slow refolding reactions. Thus the fast refolding reaction is UF leads to N and the slow refolding reaction is Us leads to N, where N is native cyt c, and there is a US in equilibrium UF equilibrium in unfolded cyt c. The results are consistent with the UF in equilibrium US reaction being proline isomerization, but this has not yet been tested in detail. Folding intermediates have been detected in both reactions. In the UF leads to N reaction, the Soret absorbance change precedes the recovery of the native 695-nm band spectrum, showing that Soret absorbance monitors the formation of a folding intermediate. In the US leads to N reaction an ascorbate-reducible intermediate has been found at an early stage in folding and the Soret absorbance change occurs together with the change at 695 nm as N is formed in the final stage of folding.     

Mössbauer studies on tetra(p-sulphophenyl)porphine iron(III) solutions

The Mössbauer spectra of frozen aqueous solutions of iron(III) over the pH range 1.9 to 12 are reported. The spectra show that tetra(p-sulphophenyl)porphine iron(III) exists principally in two forms, monomeric and as μ-oxo-oligomers, depending on the pH of the solution. Above pH 4.0 only the μ-oxo-oligomer exists and at very low pHs (2.0 or less) only monomers are present. The quadrupole splitting of the monomer is the largest yet recorded for a five coordinate high spin iron(III) porphyrin.     

Complexes of Fe(III) with amino sugars and small glycopeptides.

The complexes of Fe(III) with small molecules (glycopeptides, amino sugars) related to peptidoglycan monomer (PGM) structure were prepared and characterized by chemical and physicochemical methods. A simple method for preparation using separation of the reaction products on a molecular sieve was introduced. Using this method, monomeric complexes of small molecular weight were obtained. The likely structures were proposed on the basis of analytical results.     

Equilibrium and kinetic studies on folding of canine milk lysozyme

Thermal and chemical unfolding studies of the calcium-binding canine lysozyme (CL) by fluorescence and circular dichroism spectroscopy show that, upon unfolding in the absence of calcium ions, a very stable equilibrium intermediate state is formed. At room temperature and pH 7.5, for example, a stable molten globule state is attained in 3 M GdnHCl. The existence of such a pure and stable intermediate state allowed us to extend classical stopped-flow fluorescence measurements that describe the transition from the native to the unfolded form, with kinetic experiments that monitor separately the transition from the unfolded to the intermediate state and from the intermediate to the native state, respectively. The overall refolding kinetics of apo-canine lysozyme are characterized by a significant drop in the fluorescence intensity during the dead time, followed by a monoexponential increase of the fluorescence with k = 3.6 s(-1). Furthermore, the results show that, unlike its drastic effect on the stability, Ca(2+)-binding only marginally affects the refolding kinetics. During the refolding process of apo-CL non-native interactions, comparable to those observed in hen egg white lysozyme, are revealed by a substantial quenching of tryptophan fluorescence. The dissection of the refolding process in two distinct steps shows that these non-native interactions only occur in the final stage of the refolding process in which the two domains match to form the native conformation.     

The dimerization of ferrihaems: II. Equilibrium and kinetic studies of mesoferrihaem dimerization

Spectrophotometric data have been determined for mesoferrihaem at several pH values and over a range of concentration covering four orders of magnitude. The data reveal a dimerization process according to the equation 2 monomer ? dimer + H⁺, analogous to earlier findings for deuteroferrihaem and protoferrihaem. The value of K (defined as $\text{K}\text{=}\text{[dimer][H}{}^{\mathrm{+}}\text{]}\text{[}\text{monomer}\text{])}{}^2\text{)}$ was found to be 6.92 · 10^?2. This is close to the value for deuteroferrihaem but much less than that for protoferrihaem. This is interpreted in terms of possible additional bonding between the delocalized electron systems in protoferrihaem dimers relative to those of mesoferrihaem and deuteroferrihaem.Rate constants for dimerization were determined by temperature-jump spectrophotometry. The pH dependence of the rate constants is explained in terms of two distinct pathways for the dimerization process. These involve either direct reaction between two undissociated monomer molecules or alternatively an initial acid dissociation of a monomer molecule followed by reaction between an undissociated and a dissociated molecule.     

Equilibrium and kinetic studies of the aggregation of porphyrins in aqueous solution.

An investigation of the behavior of protoporphyrin IX, deuteroporphyrin IX, haematoporphyrin IX and coproporphyrin III in aqueous solution revealed extensive and complex aggregation processes. Protoporphyrin appears to be highly aggregated under all conditions studied. At concentrations below 4 muM, aggregation of deutero-, haemato- and coproporphyrin is probably restricted to dimerization. At approx. 4muM each of these three porphyrins exhibits sharp changes in spectra consistent with a "micellization" process to form large aggregates of unknown size. This critical concentration increases with increasing temperature and pH, but is not very sensitive to variation in ionic strength. Temperature-jump kinetic studies on deuteroporphyrin also imply an initial dimerization process, the rate constants for which are comparable with those for various synthetic porphyrins, followed by a further extensive aggragation. The ability of a particular porphyrin to dimerize appears to parallel that of the corresponding iron(III) complexes (ferrihaems), although it is thought that ferrihaems do not exhibit further aggregation under these conditions.