Iron(III) complexes of chrysobactin,the siderophore ofErwinia chrysanthemi

The phytopathogenic bacteriumErwinia chrysanthemi produces the monocatecholate siderophore chrysobactin under conditions of iron deprivation. Only the catecholate hydroxyl groups participate in metal coordination, and chrysobactin is therefore unable to provide full 1:1 coordination of Fe(III). The stoichiometry in aqueous solution is a variable dependent on pH and metal/ligand ratio, in addition to being concentration dependent. At neutral pH and concentrations of about 0.1mm, ferric chrysobactin exists as a mixture of bis and tris complexes. Chrysobactin and its isomers form optically active tris complexes. The dominant configuration depends on the chirality of the amino acid to which the catecholate moiety is attached.     

Iron(III) complexes of certain meridionally coordinating tridentate ligands as models for non-heme iron enzymes: the role of carboxylate coordination

The iron(III) complexes [Fe(pda)Cl(H(2)O)(2)] (1), [Fe(tpy)Cl(3)] (2), and [Fe(bbp)Cl(3)] (3), where H(2)pda is pyridine-2,6-dicarboxylic acid, tpy is 2,2':6,2'-terpyridine and bbp is 2,6-bis(benzimidazolyl)pyridine, have been isolated and studied as functional models for the intradiol-cleaving catechol dioxygenase enzymes. Mixed ligand complexes of H(2)pda with the bidentate ligands 2,2'-bipyridine (bpy) and 1,10-phenanthroline (phen) have been also prepared and studied. All the complexes have been characterized using absorption spectral and electrochemical methods. The spectral changes in the catecholate adducts of the complexes generated in situ have been investigated. Upon interacting the complexes with catecholate anions a low energy catecholate to iron(III) charge transfer band appears, which is similar to that observed for enzyme-substrate complexes. All the complexes catalyze the oxidative intradiol cleavage of 3,5-di-tert-butylcatechol (H(2)dbc) in the presence of dioxygen. Interestingly, on replacing the pyridyl groups in 2 and the bulky benzimidazole groups in 3 by the carboxylate groups, the yields of the intradiol cleavage products of dioxygenation increases, 1 (50%)>2 (20%)>3 (10%). The higher intradiol yield for 1 has been ascribed to the meridional coordination of two carboxylate groups of pda(2-). In contrast to the trend in the intradiol cleavage yields, a tremendous decrease in the rate (200 times) is observed on replacing the two pyridyl moieties in 2 by two carboxylates as in 1 and a significant decrease in rate is observed on replacing the pyridyl moieties in 2 by strongly sigma-donating benzimidazole moieties as in 3. This is in conformity with the decrease in Lewis acidities of the iron(III) centers.     

Synthesis and characterization of iron(llI) chelating analogues of siderophores on organic solid supports

A series of iron(III)-selective chelating resins have been modeled after the structural features of the naturally occurring siderophore compounds with hydroxamate, catecholate and salicylate iron binding groups. Amberlite IRC-50 was derivatized via an acid chloride intermediate to produce poly(hydroxamic acid) (IRC-50 PHA/N-H and IRC-50 PHA/N- CH₃), poly(catecholate) (IRC-50 PEDA 2,3-DHBAD and IRC-50 PEDA 3,4-DHBA) and poly(salicylate) (IRC-50 PEDA 2-HBAD) chelating resins with enhanced iron(III) binding capacity. The poly(hydroxamic acid) IRC-50 PHA/N-CH₃ was produced in 57% yield, the highest conversion yet reported for such derivatizations. This is the first report of a catechol or salicylate derivatization of Amberlite IRC-50. The highest overall iron(III) binding capacities yet reported for poly(hydroxamic acid) modifications of commercially available polymer supports were obtained for IRC-50 PHA/N-H (1.75 mmol Fe/g dry resin) and IRC-50 PHA/N-CH₃ (1.52 mmol Fe/g dry resin). IRC-50 PHA/N-H was also found to be an effective iron chelator when tested at ambient environmental conditions. Selectivity for iron- (III) was also determined by measuring Fe³⁺ binding capacity in the presence of Ca²⁺. UVVis spectroscopy with photoacoustic detection was used to assign the coordination environment of iron(III) in these chelating resins as bis-(FeL₂) and tris-(FeL₃) chelates. As an alternative synthetic approach, a poly(amidoxime) (PAO) chelating resin was synthesized by polymerization of appropriate monomers. Comparison between the two synthetic approaches showed that PAO bound a greater amount of iron(III) at flow-through column conditions, while IRC-50 PHA/N-H exhibited a greater iron(III) binding capacity at batch equilibration conditions.     

Structural studies on manganese(III) and manganese(IV) complexes of tetrachlorocatechol and the catalytic reduction of dioxygen to hydrogen peroxide

The mononuclear complexes (Bu₄N)[Mn(Cl₄Cat)₂(H₂O)(EtOH)] and (Bu₄N)₂[Mn(Cl₄Cat)₃] (H₂Cat=1,2-dihydroxybenzene) have been synthesised and characterised by X-ray diffraction. This work provides a direct, independent, synthesis of these complexes and an interesting example of how solvent effects can promote the formation of either a manganese(III) or manganese(IV) complex of the same ligand. The characterisation of (Bu₄N)[Mn(Cl₄Cat)₂(H₂O)(EtOH)] supports previous work that manganese(III) is extremely reluctant to form tris (catecholato) complexes due to the short `bite distance' of catecholate oxygen atoms (2.79 Å) which are unable to span the elongated coordination axes of the Jahn-Teller distorted Mn(III) ion and explains the 2:1 and 3:1 tetrachlorocatechol:manganese ratios in the Mn(III) and Mn(IV) complexes, respectively. Hydrogen peroxide production using dioxygen and hydroxylamine as substrates in acetonitrile/water mixtures, under ambient conditions, can be demonstrated with both complexes, suggesting that neither labile coordination sites nor the oxidation state of the manganese are important to the catalytic system. Turn over frequencies (TOF, moles of H₂O₂ per moles of manganese per hour) of ∼10 000 h⁻¹ are obtained and this compares very favourably with the commercial production of hydrogen peroxide by the autoxidation of 2-ethylanthrahydroquinone (AO process).     

Structures and electronic properties of the catecholatoiron complexes in relation to catechol dioxygenases: chlorocatecholatoiron complexes are compared to the 3,5-di-tert-butylcatecholatoiron complex in the solid state and in solution

Chlorocatecholatoiron complexes, [Fe(TPA)(4Cl[bond]Cat)]BPh(4) and [Fe(TPA)(3Cl[bond]Cat)]BPh(4), (4Cl[bond]Cat and 3Cl[bond]Cat: 4- and 3-chlorocatecholates, respectively; TPA: tris(2-pyridylmethyl)amine) were isolated as intermediates for the oxygenative cleavage of chlorocatechols by nonheme iron complexes. Geometric structures of these complexes together with [Fe(TPA)(DTBC)]BPh(4) (DTBC: 3,5-di-tert-butylcatecholate) as reference were analyzed by X-ray absorption spectroscopy (EXAFS) in the solid state and in solution. Structure of the DTBC complex in the solid state was shown to be noticeably different from the other complexes as seen in the magnetic susceptibility and spectroscopic data. Electronic and magnetic properties of these complexes were studied by X-ray absorption (XANES), electronic (VIS) and ESR spectroscopies, and magnetic susceptibility. Electron transfer from the catecholate ligand to the Fe(III) center was indicated by the Fe[bond]K edge values in XANES spectra and by the LMCT bands in electronic spectra. Magnetic susceptibility and ESR data indicated that at low temperatures the complexes are in equilibrium between the low (S=1/2) and high-spin (S=5/2) ferric states with the latter component increasing with temperature. Remarkable differences between the spin states in solid and in solution were observed with the DTBC complex.     

Solution equilibrium studies on metal complexes of 2,3-dihydroxy-phenylalanine-hydroxamic acid (Dopaha) and models: catecholate versus hydroxamate coordination in iron(III)-, aluminium(III)- and molybdenum(VI)-Dopaha complexes

Equilibrium results based on pH potentiometric, spectrophotometric and (1)H NMR measurements for the complexes of Fe(III), Al(III) and Mo(VI) with 2,3-dihydroxy-phenylalanine-hydroxamic acid (Dopaha) as well as for binary model systems Fe(III)-, Al(III)-, Mo(VI)-acetohydroxamic acid (Aha), -alpha-alaninehydroxamic acid (alpha-Alaha) and -1,2-dihydroxy-3,5-benzene-disulphonate (Tiron) and ternary model systems Fe(III)-, Al(III)-, Mo(VI)-Tiron-Aha, are summarized in this paper. The amine-type coordination mode is not detectable with these metal ions at all. Precipitation occurs at pH <5.5 with Fe(III) and Al(III) even at a Dopaha-to-metal ion ratio of 10:1. Hydroxamate-type coordination was demonstrated with both metals below the pH range of precipitation but, after dissolution, catecholate-type coordination was exclusively found. The hydroxamate-type coordination mode occurs only in the very acidic pH range for Mo(VI) complexes and the crossover from hydroxamate to catecholate binding occurs at pH >3. A ligand-bridged dinuclear species, [(MoO(2))(2)(Dopaha)(2)](2+), involving mixed-type (catecholate and hydroxamate) coordination modes is formed in the pH range 2.5-5.5. [MoO(2)A(2)H(2)], with catecholate-type coordination, forms above pH 3. On increasing the pH further, deprotonation of the coordinated Dopaha and hydrolytic processes result in the formation of catecholate-coordinated [MoO(3)AH] and [MoO(3)A]. MoO(4)(2-) and free Dopaha exist above pH 10.     

Synthesis, siderophore activity and iron(III) chelation chemistry of a novel mono-hydroxamate, bis-catecholate siderophore mimic: N(alpha),-N(epsilon)-Bis[2,3-dihydroxybenzoyl]-l-lysyl-(gamma-N-methyl-N-hydroxyamido)-L-glutamic acid

The synthesis and characterization of a novel tripodal mono-hydroxamate, bis catecholate siderophore mimic, N(alpha),-N(epsilon)-bis[2,3-dihydroxybenzoyl]-l-lysyl-(gamma-N-methyl-N-hydroxyamido)-l-glutamic acid (H(6)L), is described. The structure of H(6)L was established by 2D NMR and mass spectrometry. The chelation chemistry of H(6)L with respect to iron(III) is characterized in aqueous solution through determination of ligand pK(a) values and iron(III) binding constants using spectrophotometric and potentiometric titration techniques. Proton dependent iron(III)-ligand equilibrium constants were determined using a model based on the sequential protonation of the iron(III)-siderophore complex. These results were used to calculate the pH dependent speciation, the overall formation constant logbeta(110) (31.4) and pM value (18.3) for H(6)L with iron(III). The ability of H(6)L to deliver the essential nutrient iron to living cells is determined through growth promotion assays using various bacterial strains.     

Equilibrium and steady state thermodynamics of active transport systems studied on simple models simulating Ca2+ transport through sarcoplasmic reticulum membranes

Equilibrium and steady state conditions of primary active transport systems are analyzed in models simulating well known characteristics of calcium transport through sarcoplasmic reticulum membranes. The model for the equilibrium simulations is a closed system with two compartments and a vectorial chemical reaction coupling Ca transport and ATP breakdown. The chemical potential difference for Ca (delta mu Ca) is calculated as a function of the total amount of Ca (Cat) and nucleotides (Nt) in the system. Results are obtained by successive approximations along the thermodynamic pathway of the reaction, up to minimizing free energy of the system, since the solution of the explicit equations cannot be obtained with computers of current precision for data within physiological ranges. delta mu Ca and [Caout] are extremely dependent on Cat and Nt for certain combinations of the variables, i.e. [Caout] can be raised from 10(-8) to 10(-6) M when Cat varies from 0.998 to 1.002 mM, therefore, the running force of the spontaneous reaction is largely shifted by tiny changes in the parameters of the system. For steady state simulations, ATP supply to the system, ADP and Pi drainage, and Ca diffusion through the barrier, are assumed. Again, conditions within physiological ranges can be found where tiny changes in Cat, the rate of ATP supply, diffusion, the ratio between the volumes of the compartments, or a relative uncoupling between the transport and hydrolytic reactions, largely shifts delta mu Ca and [Caout], thus making the steady state highly unstable and therefore well designed to operate as an amplifier of physiological signals. The equilibrium model describes some physicochemical characteristics of the system; the steady state model is more useful to simulate several physiological situations.     

Kinetics and mechanism of dissimilative Fe(III) reduction by Pseudomonas sp. 200

The kinetics and mechanism of Fe(III) reduction to Fe(II) were studied in pure batch cultures of Pseudomonas sp. 200. The rate of iron reduction has been mechanistically related to aqueous phase iron speciation. In the absence of microbial activity the iron reduction rate was negligible. Initial rates of microbial iron reduction were accelerated more than 20-fold by the addition of equimolar quantities of nitrilotriacetic acid (NTA) to media initially containing 1.86 x 10(-3)M total Fe(III). Numerical techniques were utilized to quantify relationships between the observed rate of Fe(II) production and the calculated (equilibrium) aqueous phase speciation. These results indicate that soluble ferric iron species are not equivalent in terms of their susceptibility to bacterial (dissimilative) iron reduction. The concentration of Fe(NTA)(OH)(2) (2-) correlated strongly with observed iron reduction rates. Ferrous iron species appeared to inhibit the reduction process.     

Abundance and phylogenetic affiliation of iron reducers in activated sludge as assessed by fluorescence in situ hybridization and microautoradiography

Microautoradiography (MAR) was used to enumerate acetate-consuming bacteria under Fe(III)-reducing conditions in activated sludge. This population is believed to consist of dissimilatory iron-reducing bacteria, because the applied incubation conditions and the use of specific inhibitors excluded consumption of radiolabeled acetate by other physiological groups such as sulfate reducers. By use of this approach, dissimilatory iron reducers were found in a concentration of 1.1 x 10(8) cells per ml, corresponding to approximately 3% of the total cell count as determined by DAPI (4',6'-diamino-2-phenylindoledihydrochloride-dilactate) staining. The MAR enumeration method was compared to the traditional most-probable-number (MPN) method (FeOOH-MPN) and a modified MPN method, which contains Ferrozine directly within the MPN dilutions to determine the production of small amounts of ferrous iron (Ferrozine-MPN). The Ferrozine-MPN method yielded values 6 to 10 times higher than those obtained by the FeOOH-MPN method. Nevertheless, the MAR approach yielded counts that were 100 to 1,000 times higher than those obtained by the Ferrozine-MPN method. Specific in situ Fe(III) reduction rates per cell (enumerated by the MAR method) were calculated and found to be comparable to the respective rates for pure cultures of dissimilatory iron-reducing bacteria, suggesting that the new MAR method is most reliable. A combination of MAR and fluorescence in situ hybridization was used for phylogenetic characterization of the putative iron-reducing bacteria. All activated-sludge cells able to consume acetate under iron-reducing conditions were targeted by the bacterial oligonucleotide probe EUB338. Around 20% were identified as gamma Proteobacteria, and 10% were assigned to the delta subclass of Proteobacteria.     

Unique iron coordination in iron-chelating molecule vibriobactin helps Vibrio cholerae evade mammalian siderocalin-mediated immune response

Iron is essential for the survival of almost all bacteria. Vibrio cholerae acquires iron through the secretion of a catecholate siderophore called vibriobactin. At present, how vibriobactin chelates ferric ion remains controversial. In addition, the mechanisms underlying the recognition of ferric vibriobactin by the siderophore transport system and its delivery into the cytoplasm specifically have not been clarified. In this study, we report the high-resolution structures of the ferric vibriobactin periplasmic binding protein ViuP and its complex with ferric vibriobactin. The holo-ViuP structure reveals that ferric vibriobactin does not adopt the same iron coordination as that of other catecholate siderophores such as enterobactin. The three catechol moieties donate five, rather than six, oxygen atoms as iron ligands. The sixth iron ligand is provided by a nitrogen atom from the second oxazoline ring. This kind of iron coordination results in the protrusion of the second catechol moiety and renders the electrostatic surface potential of ferric vibriobactin less negatively polarized compared with ferric enterobactin. To accommodate ferric vibriobactin, ViuP has a deeper subpocket to hold the protrusion of the second catechol group. This structural characteristic has not been observed in other catecholate siderophore-binding proteins. Biochemical data show that siderocalin, which is part of the mammalian innate immune system, cannot efficiently sequester ferric vibriobactin in vitro, although it can capture many catecholate siderophores with high efficiency. Our findings suggest that the unique iron coordination found in ferric vibriobactin may be utilized by some pathogenic bacteria to evade the siderocalin-mediated innate immune response of mammals.     

The kinetics and mechanisms of the reaction of iron(III) with gallic acid, gallic acid methyl ester and catechin.

The kinetics and mechanisms of the reactions of a number of pyrogallol-based ligands with iron(III) have been investigated in aqueous solution at 25 degrees C and ionic strength 0.5 M NaClO(4). Mechanisms have been proposed which account satisfactorily for the kinetic data. These are generally consistent with a mechanism in which the 1:1 complex that is formed initially when the metal reacts with the ligand subsequently decays through an electron transfer reaction. There was also some evidence for the formation of a 1:2 ligand-to-metal complex at higher pH values. The kinetics of complex formation were investigated with either the ligand or metal in pseudo-first-order excess. Rate constants for k(1) of 2.83(+/-0.09)x10(3), 1.75(+/-0.045)x10(3) and 3300(+/-200) M(-1) s(-1) and k(-1) of 20(+/-6.0), 35(+/-13) and 25+/-7.6 M(-1) s(-1) have been evaluated for the reaction of Fe(OH)(2+) with gallic acid, gallic acid methyl ester and catechin, respectively. The stability constant of each [Fe(L)](+) complex has been calculated from the kinetic data. The iron(III) assisted decomposition of the initial iron(III) complex formed was investigated. Analysis of the kinetic data yielded both the equilibrium constants for protonation of the iron(III) complexes initially formed together with the rate constants for the intramolecular electron transfers for gallic acid and gallic acid methyl ester. All of the suggested mechanisms and calculated rate constants are supported by calculations carried out using global analysis of time-dependent spectra.     

Abundance, distribution, and activity of Fe(II)-oxidizing and Fe(III)-reducing microorganisms in hypersaline sediments of Lake Kasin, southern Russia

The extreme osmotic conditions prevailing in hypersaline environments result in decreasing metabolic diversity with increasing salinity. Various microbial metabolisms have been shown to occur even at high salinity, including photosynthesis as well as sulfate and nitrate reduction. However, information about anaerobic microbial iron metabolism in hypersaline environments is scarce. We studied the phylogenetic diversity, distribution, and metabolic activity of iron(II)-oxidizing and iron(III)-reducing Bacteria and Archaea in pH-neutral, iron-rich salt lake sediments (Lake Kasin, southern Russia; salinity, 348.6 g liter(-1)) using a combination of culture-dependent and -independent techniques. 16S rRNA gene clone libraries for Bacteria and Archaea revealed a microbial community composition typical for hypersaline sediments. Most-probable-number counts confirmed the presence of 4.26 × 10(2) to 8.32 × 10(3) iron(II)-oxidizing Bacteria and 4.16 × 10(2) to 2.13 × 10(3) iron(III)-reducing microorganisms per gram dry sediment. Microbial iron(III) reduction was detected in the presence of 5 M NaCl, extending the natural habitat boundaries for this important microbial process. Quantitative real-time PCR showed that 16S rRNA gene copy numbers of total Bacteria, total Archaea, and species dominating the iron(III)-reducing enrichment cultures (relatives of Halobaculum gomorrense, Desulfosporosinus lacus, and members of the Bacilli) were highest in an iron oxide-rich sediment layer. Combined with the presented geochemical and mineralogical data, our findings suggest the presence of an active microbial iron cycle at salt concentrations close to the solubility limit of NaCl.     

Unprecedented forms of vanadium observed within the blood cells of Phallusia nigra using K-edge X-ray absorption spectroscopy

Fits to the vanadium K-edge X-ray absorption spectra (XAS) of five whole blood cell samples from the tunicate Phallusia nigra revealed unprecedented forms of intracellular vanadium. Endogenous vanadium was divided between the V(III) ion (74.2+/-5.1% of total V) and the vanadyl ion [V(IV)=O](2+) (25.2+/-5.4% of total V). The V(III) fraction included both [V(H(2)O)(6)](3+) (36.7+/-5.5%) modeled as VCl(3) in 1 M HCl, and three previously unprecedented chelated V(III) forms (37.5+/-4.6%). Two of these could be represented by the model ligand environments V(acetylacetonate)(3) (17.9+/-3.2%) and K(3)V(catecholate)(3) (13.1+/-4.7%), implying DOPA-like complexation. The third chelated form was represented by the 7-coordinate N(2)O(5) complex Na[V(edta)(H(2)O)] (8.0+/-1.8%). This coordination array, suggestive of a novel mononuclear V(III) protein site, contributed only to fits to samples 1, 2, 3 and 5, which were prepared in the presence of DTT. Endogenous V(IV) (25.2+/-5.4%) was principally modeled as VOCl(2) in 1 M HCl. EPR spectra (averages: A(parallel)=(1.842+/-0.006)x10(-2) cm(-1); A( perpendicular)=(0.718+/-0.007)x10(-2) cm(-1); g(parallel)=1.936+/-0.002; g( perpendicular)=1.990+/-0.001) confirmed the predominance of the aquated vanadyl ion. Blood cell sample five uniquely required the XAS spectrum of VOSO(4) in 0.1 M H(2)SO(4) solution (13.0%) and of [OV(V)(pivalate)(3)] (3.1%) to successfully fit the XAS pre-edge energy region. This endogenous V(V) signal is also unprecedented. These results are compared with those of analogous fits to the blood cells of Ascidia ceratodes and may support assignment of P. nigra to a different genus.     

Ferric iron complexes of dopamine and 5,6-dihydroxyindole with nta, edda, and edta as ancillary ligands

Coordination of the neurotransmitter dopamine (DA) and the metal-binding component of neuromelanin 5,6-dihydroxyindole (DHI) with ferric iron has been studied in aqueous solution in the presence of ancillary ligands containing amine nitrogen and carboxylate oxygen donor sites. With nitrilotriacetic acid (nta) and ethylenediamine diacetic acid (edda) coligands, coordination of the catecholate ligands DA and DHI is observed to be complete at physiological pH. The resulting complexes of DA have the characteristic two-component electronic spectrum observed characteristically for L₄Fe(Cat) complexes. The spectrum obtained with DHI consists of a single broad absorption in the visible region. Both DA and DHI are able to coordinate with Fe³⁺ in the presence of edta, displacing carboxylate oxygen donors at pH values just above physiological pH. These results demonstrate the strong affinity of DA and DHI for Fe³⁺, pointing to in vivo complex formation in neuronal mixtures at physiological pH.     

EXAFS studies of Fe(III)-phosvitin at high metal to protein ratios

The stereochemistry of the Fe(III) binding sites in chicken egg phosvitin (PST) at very high iron content, in solution and as a powder, has been investigated through EXAFS spectroscopy. We found that the EXAFS spectra obtained for aqueous PST solutions at metal:protein ratios of 20:1 and 40:1 are very similar to those previously obtained by us on a Fe₁₀PST sample. In all cases the iron ions are octahedrally coordinated by oxygen atoms of the serine-bound phosphate groups and by other ligands from either the protein or the solvent. The average metal-donor atom distance is 1.94 Å. At variance, the EXAFS results for a Fe₅₀PST powder sample suggest the occurrence of a switch in iron coordination from octahedral to lower coordination numbers (5,4). The average iron-oxygen distance is virtually unchanged; apparently, four iron ligands are provided by four different coordinate phosphate groups from the phosphorylated serine residues abundant in the protein. This finding contains interesting implications for the structure-function relationships of this intriguing protein.     

Resonance Raman studies on the blue-green-colored bovine adrenal tyrosine 3-monooxygenase (tyrosine hydroxylase). Evidence that the feedback inhibitors adrenaline and noradrenaline are coordinated to iron

Tyrosine 3-monooxygenase (tyrosine hydroxylase) is a non-heme iron, tetrahydropterin-dependent enzyme which catalyzes the rate-limiting step in the biosynthesis of catecholamines. The highly purified bovine adrenal enzyme contains an unusual blue-green chromophore with lambda max at around 700 nm (epsilon = 1.3 (mM subunit enzyme)-1 cm-1). On excitation at 605.2 nm, resonance-enhanced Raman vibrations are observed at 454, 494, 527, 604, 635, 835, 1130, 1271, 1320, 1426, and 1476 cm-1. The excitation profiles of the modes of 1276 and 1476 cm-1 (from 488 to 620 nm) follow the contour of the 700 nm absorption band. The vibrations observed strongly indicate the presence of a bidentate catecholamine-Fe(III) complex in the enzyme as isolated which gives rise to the characteristic charge-transfer transitions. This is further supported by the release of 0.11 +/- 0.04 mol of noradrenaline and 0.25 +/- 0.06 mol of adrenaline per mol of enzyme subunit on denaturation of the enzyme. The energies of the catecholate to Fe(III) charge-transfer transitions indicate a mixture of histidines and carboxylate(s) coordinated to the iron center in tyrosine hydroxylase. At neutral pH, the enzymatic activity was inhibited more than 50% by 10 microM dopamine, noradrenaline, and adrenaline. The high affinity of the catecholamines to the nonphosphorylated form of tyrosine hydroxylase may have significance in vivo since catecholamines are potent feedback inhibitors of the enzyme.     

The kinetics and mechanisms of the complex formation and antioxidant behaviour of the polyphenols EGCg and ECG with iron(III)

(-)-Epigallocatechin-gallate ((-)-EGCg) and (-)-epicatechin-gallate ((-)-ECG) are important antioxidants which are found in green tea. The kinetics and mechanisms of the reactions of a pseudo-first order excess of iron(III) with EGCg and ECG have been investigated in aqueous solution at 25 degrees C and an ionic strength of 0.5M NaClO(4). Mechanisms have been proposed which account satisfactorily for the kinetic data. These are consistent with a mechanism in which the 2:1 metal:ligand complex initially formed on reaction of iron(III) with the ligand subsequently decomposes in an electron transfer step. Complex formation takes place at two separate binding sites via coupled reactions. Rate constants of 4.28(+/-0.06) x 10(6) M(-2) s(-1) and 2.83(+/-0.04) x 10(6) M(-2) s(-1) have been evaluated for the reaction of monohydroxy Fe(OH)2+ species with EGCg and ECG, respectively while rate constants for of 2.94(+/-0.4) x 10(4) M(-2) s(-1) and 2.41(+/-0.25) x 10(4) M(-2) s(-1) have been evaluated for the reaction of Fe3+ species with EGCg and ECG, respectively. 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 EGCg and ECG 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. The results obtained show that one molecule of either EGCg or EGC is capable of reducing up to four iron(III) species, a fact which is consistent with the powerful antioxidant properties of the ligands.     

Nitrogenase X: M?ssbauer and EPR studies on reversibly oxidized MoFe protein from Azotobacter vinelandii OP. Nature of the iron centers.

Under anaerobic conditions the molybdenum-iron protein (MoFe protein) from Azotobacter vinelandii can be reversibly oxidized with thionine. Electron paramagnetic resonance studies reveal that the oxidation proceeds in two distinct phases: the MoFe protein can be oxidized by four electrons without loss of the EPR signal from the S = 3/2 cofactor centers. A second oxidation step, involving two electrons, leads to the disappearance of the cofactor EPR signal. In order to correlate the events during the thionine titration with redox reactions involving individual iron centers we have studied the MoFe proteins from A vinelandii and Clostridium pasteurianum with M?ssbauer spectroscopy. Spectra were taken in the temperature range from 1.5 K to 200 K in applied magnetic fields of up to 54 kG. Analysis of the M?ssbauer data allows us to draw three major conclusions: (1) the holoprotein contains 30 +/- 2 iron atoms. (2) Most probably, 12 iron atoms belong to two, apparently identical, iron clusters (labeled M) which we have shown previously to be structural components of the iron and molybdenum containing cofactor of nitrogenase. The M-centers can be stabilized in three distinct oxidation states, MOXe- in equilibrium MNe- in equilibrium MR. The diamagnetic (S = 0) state MOX is attained by oxidation of the native state MN with either thionine or oxygen. MR is observed under nitrogen fixing conditions. (3) The data strongly suggest that 16 iron atoms are associated with four iron centers which we propose to call P-clusters. Each P-cluster contains four spin-coupled iron atoms. In the native protein the P-clusters are in the diamagnetic state PN, yielding the M?ssbauer signature which we have labeled previously 'components D and Fe2+'. Three irons of the D-type and one iron of the Fe2+-type appear to comprise a P-cluster. A one-electron oxidation yields the paramagnetic state POX. Although the state POX is characterized by half-integral electronic spin a peculiar combination of zero-field splitting parameters and spin relaxation renders this state EPR-silent. Spectroscopically, the P-clusters are novel structures; there is, however, evidence that they are closely related to familiar 4Fe-4S centers.