O2-binding albumin thin films: solid membranes of poly(ethylene glycol)-conjugated human serum albumin incorporating iron porphyrin

Poly(ethylene glycol) (PEG)-conjugated human serum albumin (HSA) incorporating the tetrakis(alpha,alpha,alpha,alpha-o-amidophenyl)porphinatoiron(II) derivative (FeP) [PEG(HSA-FeP)] is a unique plasma protein-based O2 carrier as a red blood cell substitute. The aqueous solution of PEG(HSA-FeP) [mw of PEG: 2-kDa (PEG2) or 5-kDa (PEG5)] was evaporated on a glass surface to produce a red-colored solid membrane. Scanning electron microscopy observations revealed that the PEG2(HSA-FeP) membrane consisted of two parts: (i) a surface layer made of a fibrous component (10 microm thickness), and (ii) a bottom layer of an amorphous phase (5 microm thickness). The condensed solution provided a thick membrane (70 microm), which also has the amorphous bottom layer. On the other hand, the PEG5(HSA-FeP) produced homogeneous membrane made of the fibrous component. The FeP active sites in the solid membrane formed very stable O2-adduct complexes at 37 degrees C with a half-lifetime of 40 h. The O2-binding affinity of the PEG2(HSA-FeP) membrane (P1/2 = 40 Torr, 25 degrees C) was 4-fold lower than that in aqueous solution, which is kinetically due to the low association rate constant. The membrane was soluble again in water and organic solvents (ethanol and chloroform) without deformation of the secondary structure of the protein. The addition of hyaluronic acid gave a free-standing flexible thin film, and it can also bind and release O2 as well. These O2-carrying albumin membranes with a micrometer-thickness would be of significant medical importance for a variety of clinical treatments.     

Exchange transfusion with albumin-heme as an artificial O2-infusion into anesthetized rats: physiological responses, O2-delivery, and reduction of the oxidized hemin sites by red blood cells

Human serum albumin (HSA) incorporating synthetic hemes, the tetrakis(o-pivalamido)phenylporphinatoiron(II) derivative (FeP), is an artificial hemoprotein (HSA-FeP) which is able to reversibly bind and release dioxygen under physiological conditions (in aqueous media, pH 7.4, 37 degrees C) like hemoglobin and myoglobin. Physiological responses to exchange transfusion with HSA-FeP solution [[HSA], 5 g/dL; FeP/HSA, 4 (mol/mol)] into rats after hemodilution and hemorrhage (Hct, about 10%) has been evaluated. The declined mean arterial pressure (MAP) and blood flow after a 70% exchange with HSA and the further 40% bleeding of blood were significantly recovered up to about 90% of the baseline values by the injection of HSA-FeP. Furthermore, the renal cortical O(2)-tensions and skeletal tissue O(2)-tensions were also increased, indicating the in vivo O(2)-delivery of HSA-FeP. Autoxidation of ferrous Fe(II)P to ferric Fe(III)P was retarded in the blood stream; the half-lifetime of the dioxygenated FeP [tau(1/2)(O(2))] in vivo was 4.1 h [cf. 1.0 h (in vitro)]. It has been found that autooxidized Fe(III)P was certainly reduced in the whole blood suspension. Physiological concentrations of ascorbic acid continuously provided by red blood cells probably rereduces Fe(III)P, leading to the apparent long lifetime of the dioxygenated species of FeP.     

Synthesis and oxidation of carboxylate-bridged diiron(II) complexes with substrates tethered to primary alkyl amine ligands

The synthesis and crystallographic characterization of a series of diiron(II) complexes with sterically hindered terphenyl carboxylate ligands and alkyl amine donors are presented. The compounds [Fe(2)(mu-O(2)CAr(Tol))(4)(L)(2)] (L=NH(2)(CH(2))(2)SBn (1); NH(2)(CH(2))(3)SMe (2); NH(2)(CH(2))(3)CCH (3)), where (-)O(2)CAr(Tol) is 2,6-di(p-tolyl)benzoate, and [Fe(2)(mu-O(2)CAr(Xyl))(2)(O(2)CAr(Xyl))(2)(L)(2)] (L=NH(2)(CH(2))(3)SMe (4); NH(2)(CH(2))(3)CCH (5)), where (-)O(2)CAr(Xyl) is 2,6-di(3,5-dimethylphenyl)benzoate, were prepared as small molecule mimics of the catalytic sites of carboxylate-bridged non-heme diiron enzymes. The compounds with the (-)O(2)CAr(Tol) carboxylate form tetrabridged structures, but those containing the more sterically demanding (-)O(2)CAr(Xyl) ligand have only two bridging ligands. The ancillary nitrogen ligands in these carboxylate-rich complexes incorporate potential substrates for the reactive metal centers. Their oxygenation chemistry was studied by product analysis of the organic fragments following decomposition. Compound 1 reacts with dioxygen to afford PhCHO in approximately 30% yield, attributed to oxidative dealkylation of the pendant benzyl group. Compound 3 decomposes to form Fe(II)Fe(III) and Fe(III)Fe(IV) mixed-valence species by established bimolecular pathways upon exposure to dioxygen at low temperatures. Upon decomposition, the alkyne-substituted amine ligand was recovered quantitatively. When the (-)O(2)CAr(Tol) carboxylate was replaced by the (-)O(2)CAr(Xyl) ligand in 5, different behavior was observed. The six-coordinate iron(III) complex with one bidentate and two monodentate carboxylate ligands, [Fe(O(2)CAr(Xyl))(3)(NH(2)(CH(2))(3)CCH)(2)] (6), was isolated from the reaction mixture following oxidation.     

Identification of the sites of deoxyhaemoglobin PEGylation

The 2-iminothiolane reaction with protein amino groups adds a spacer arm ending with a thiol group, which can be further treated with molecules carrying a maleimido ring. This approach is currently used for the preparation of a candidate 'blood substitute' in which human Hb (haemoglobin) is conjugated with long chains of PEG [poly(ethylene glycol)]. To identify the thiolation sites by MS, we have carried out the reaction using deoxyHb bound to inositol hexaphosphate to protect some of the residues crucial for function and NEM (N-ethylmaleimide) to block and stabilize the thiol groups prior to enzymatic digestion by trypsin and pepsin. Under the conditions for the attachment of 5-8 PEG chains per tetramer, the thiolated residues were Lys7, Lys11, Lys16, Lys56 and Lys139 and, with lower accessibility, Lys90, Lys99 and Lys60 of the a-chain and Lys8, Lys17, Lys59, Lys61 and Lys66 and, with lower accessibility, Lys65, Lys95 and Lys144 of the b-chain. The a-amino groups of a- and b-chains were not modified and the reaction of the Cysb93 residues with NEM was minor or absent. After the modification with thiolane and NEM of up to five to eight lysine residues per tetramer, the products retained a large proportion of the properties of native Hb, such as low oxygen affinity, co-operativity, effect of the modulators and stability to autoxidation. Under identical anaerobic conditions, the conjugation of the thiolated Hb tetramer with five or six chains of the maleimido derivative of 6 kDa PEG yielded products with diminished co-operativity, Hill coefficient h=1.3-1.5, still retaining a significant proportion of the effects of the modulators of oxygen affinity and stability to autoxidation. Co-operativity was apparently independent of the topological distribution of the PEGylated sites as obtained by treating partly the thiolated protein with NEM prior to PEGylation [poly(ethylene glycol)ation].     

Ferrocyanide - a novel catalyst for oxymyoglobin oxidation by molecular oxygen

A comparative study of the rates of ferrocyanide-catalyzed oxidation of several oxymyoglobins by molecular oxygen is reported. Oxidation of the native oxymyoglobins from sperm whale, horse and pig, as well as the chemically modified (MbO(2)) sperm whale oxymyoglobin, with all accessible His residues alkylated by sodium bromoacetate (CM-MbO(2)), and the mutant sperm whale oxymyoglobin [MbO(2)(His119-->Asp)], was studied. The effect of pH, ionic strength and the concentration of anionic catalyst ferrocyanide, [Fe(CN)(6)](4-), on the oxidation rate is investigated, as well as the effect of MbO(2) complexing with redox-inactive Zn(2+), which forms the stable chelate complex with functional groups of His119, Lys16 and Asp122, all located nearby. The catalytic mechanism was demonstrated to involve specific [Fe(CN)(6)](4-) binding to the protein in the His119 region, which agrees with a high local positive electrostatic potential and the presence of a cavity large enough to accommodate [Fe(CN)(6)](4-) in that region. The protonation of the nearby His113 and especially His116 plays a very important role in the catalysis, accelerating the oxidation rate of bound [Fe(CN)(6)](4-) by dissolved oxygen. The simultaneous occurrence of both these factors (i.e. specific binding of [Fe(CN)(6)](4-) to the protein and its fast reoxidation by oxygen) is necessary for the efficient ferrocyanide-catalyzed oxidation of oxymyoglobin.     

Direct observation of covalent adducts with Cys34 of human serum albumin using mass spectrometry

The interactions of the unpaired thiol residue (Cys34) of human serum albumin (HSA) with low-molecular-weight thiols and an Au(I)-based antiarthritic drug have been examined using electrospray ionization mass spectrometry. Early measurements of the amount of HSA containing Cys34 as the free thiol suggested that up to 30% of circulating HSA bound cysteine as a mixed disulfide. It has also been suggested that reaction of HSA with cysteine, occurs only on handling and storage of plasma. In our experiments, there were three components of HSA in freshly collected plasma from normal volunteers, HSA, HSA+cysteine, and HSA+glucose in the ratio approximately 50:25:25. We addressed this controversy by using iodoacetamide to block the free thiol of HSA in fresh plasma, preventing its reaction with plasma cysteine. When iodoacetamide was injected into a vacutaner tube as blood was collected, the HSA was modified by iodoacetamide, with 20-30% present as the mixed disulfide with cysteine (HSA+cys). These data provide strong evidence that 20-30% of HSA in normal plasma contains one bound cysteine. Reaction of HSA with [Au(S(2)O(3))(2)](3-) resulted in formation of the adducts HSA+Au(S(2)O(3)) and HSA+Au. Reaction of HSA with iodoacetamide prior to treatment with [Au(S(2)O(3))(2)](3-) blocked the formation of gold adducts.     

Reactions of peroxyl radicals with Fe(H(2)O)(6)(2+)

The reactions of RO(2)* radicals with Fe(H(2)O)(6)(2+) were studied, R[double bond]H; CH(3); CH(2)COOH; CH(2)CN; CH(2)C(CH(3))(2)OH; CH(2)OH; CHCl(2)/CCl(3). All these processes involve the following reactions: Fe(H(2)O)(6)(2+)+RO(2)*<==>(H(2)O)(5)Fe(III)[bond]OOR(2+) K(1) approximately 250 M(-1); (H(2)O)(5)Fe(III)[bond]OOR(2+)+H(3)O(+)/H(2)O-->Fe(H(2)O)(6)(3+)+ROOH+H(2)O/OH(-); (H(2)O)(5)Fe(III)[bond]OOR(2+)+2Fe(H(2)O)(6)(2+)-->3Fe(H(2)O)(6)(3+)+ROH; 2 RO(2)*-->Products; RO(2)*+(H(2)O)(5)Fe(III)[bond]OOR(2+)-->Fe(H(2)O)(6)(2+)+products. The values of k(1) and k(3) [reaction is clearly not an elementary reaction] approach the ligand exchange rate of Fe(H(2)O)(6)(2+), i.e. these reactions follow an inner sphere mechanism and the rate determining step is the ligand exchange step. The rate of reaction is several orders of magnitude faster than that of the Fenton reaction. Surprisingly enough the K(1) values are nearly independent of the redox potential of the radical and are considerably higher than calculated from the relevant redox potentials. These results indicate that the ROO(-) ligands considerably stabilise the Fe(III) complex, this stabilisation is smaller for radicals with electron withdrawing groups which raise the redox potential of the radical but decrease the basicity of the ROO(-) ligands, two effects which seem to nearly cancel each other. Finally, the results clearly indicate that reaction (5) is relatively fast and affects the nature of the final products. The contribution of these reactions to oxidation processes involving 'Fenton-like' processes is discussed.     

Identification of the interactions between cytochrome P450 2E1 and cytochrome b5 by mass spectrometry and site-directed mutagenesis

The reaction cycles of cytochrome P450s (P450) require input of two electrons. Electrostatic interactions are considered important driving forces in the association of P450s with their redox partners, which in turn facilitates the transfer of the two electrons. In this study, the cross-linking reagent, 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC), was used to covalently link cytochrome P450 2E1 (CYP2E1) with cytochrome b(5) (b(5)) through the formation of specific amide bonds between complementary charged residue pairs. Cross-linked peptides in the resulting protein complex were distinguished from non-cross-linked peptides using an (18)O-labeling method on the basis that cross-linked peptides incorporate twice as many (18)O atoms as non-cross-linked peptides during proteolysis conducted in (18)O-water. Subsequent tandem mass spectrometric (MS/MS) analysis of the selected cross-linked peptide candidates led to the identification of two intermolecular cross-links, Lys(428)(CYP2E1)-Asp(53)(b(5)) and Lys(434)(CYP2E1)-Glu(56)(b(5)), which provides the first direct experimental evidence for the interacting orientations of a microsomal P450 and its redox partner. The biological importance of the two ion pairs for the CYP2E1-b(5) interaction, and the stimulatory effect of b(5), was confirmed by site-directed mutagenesis. Based on the characterized cross-links, a CYP2E1-b(5) complex model was constructed, leading to improved insights into the protein interaction. The described method is potentially useful for mapping the interactions of various P450 isoforms and their redox partners, because the method is relatively rapid and sensitive, and is capable of suggesting not only protein interacting regions, but also interacting orientations.     

The role of photoinduced electron transfer processes in photodegradation of the [Fe4(μ3-S)3(NO)7] cluster

Spectroscopic and electrochemical study of the [Fe(4)(mu(3)-S)(3)(NO)(7)](-) photochemical reaction and thermodynamic calculations of relevant systems demonstrate the redox character of this process. The photoinduced electron transfer between substrate clusters in excited and ground state (probably via exciplex formation) results in dismutation yielding unstable [Fe(4)(mu(3)-S)(3)(NO)(7)](2-) and [Fe(4)(mu(3)-S)(3)(NO)(7)](0). Back electron transfer between the primary products is responsible for fast reversibility of the photochemical reaction in deoxygenated solutions. In the presence of an electron acceptor (such as O(2), MV(2+) or NO) an oxidative quenching of the (*)[Fe(4)(mu(3)-S)(3)(NO)(7)](-) is anticipated, although NO seems to participate as well in the reductive quenching. The electron acceptors can also regenerate the substrate from its reduced form ([Fe(4)(mu(3)-S)(3)(NO)(7)](2-)), whereas the other primary product ([Fe(4)(mu(3)-S)(3)(NO)(7)](0)) decomposes to the final products. The suggested mechanism fits well to all experimental observations and shows the thermodynamically favored pathways and explains formation of all major (Fe(2+), S(2-), NO) and minor products (N(2)O, Fe(3+)). The photodissociation of nitrosyl ligands suggested earlier as the primary photochemical step cannot be, however, definitely excluded and may constitute a parallel pathway of [Fe(4)(mu(3)-S)(3)(NO)(7)](-) photolysis.     

Lysine biosynthesis in Saccharomyces cerevisiae: mechanism of alpha-aminoadipate reductase (Lys2) involves posttranslational phosphopantetheinylation by Lys5.

A key step in fungal biosynthesis of lysine, enzymatic reduction of alpha-aminoadipate at C6 to the semialdehyde, requires two gene products in Saccharomyces cerevisiae, Lys2 and Lys5. Here, we show that the 31-kDa Lys5 is a specific posttranslational modification catalyst, using coenzyme A (CoASH) as a cosubstrate to phosphopantetheinylate Ser880 of the 155-kDa Lys2 and activate it for catalysis. Lys2 was subcloned from S. cerevisiae and expressed in and purified from Escherichia coli as a full-length 155-kDa enzyme, as a 105-kDa adenylation/peptidyl carrier protein (A/PCP) fragment (residues 1-924), and as a 14-kDa PCP fragment (residues 809-924). The apo-PCP fragment was covalently modified to phosphopantetheinylated holo-PCP by pure Lys5 and CoASH with a Km of 1 microM and kcat of 3 min-1 for both the PCP and CoASH substrates. The adenylation domain of the A/PCP fragment activated S-carboxymethyl-L-cysteine (kcat/Km = 840 mM-1 min-1) at 16% the efficiency of L-alpha-aminoadipate in [32P]PPi/ATP exchange assays. The holo form of the A/PCP 105-kDa fragment of Lys2 covalently aminoacylated itself with [35S]S-carboxymethyl-L-cysteine. Addition of NADPH discharged the covalent acyl-S-PCP Lys2, consistent with a reductive cleavage of the acyl-S-enzyme intermediate. These results identify the Lys5/Lys2 pair as a two-component system in which Lys5 covalently primes Lys2, allowing alpha-aminoadipate reductase activity by holo-Lys2 with catalytic cycles of autoaminoacylation and reductive cleavage. This is a novel mechanism for a fungal enzyme essential for amino acid metabolism.     

Interaction of a ruthenium hexacationic prism with amino acids and biological ligands: ESI mass spectrometry and NMR characterisation of the reaction products

Reactions between the cationic triangular metallaprism [(p-cymene)(6)Ru(6)(tpt)(2)(dhnq)(3)](6+) ([1](6+)) [tpt?is?2,4,6-tri(pyridine-4-yl)-1,3,5-triazine; dhnq?is?5,8-dihydroxy-1,4-naphthoquinonato] and Arg, His, Lys, ascorbic acid, lactic acid and glutathione (GSH) have been studied at 37?°C in aqueous solution at pD 7 using NMR spectroscopy and electrospray ionisation mass spectrometry. Coordination to the imidazole nitrogen atom of His or to the basic NH/NH(2) groups in Arg and Lys slowly displaces the dhnq and tpt ligands from the (p-cymene)Ru units, and subsequently additional coordination to the amino and carboxylato groups forms stable N,N,O metallacycles. Compared with our previously reported study with the analogous metallaprism [(p-cymene)(6)Ru(6)(tpt)(2)(dhbq)(3)](6+) ([2](6+)) (dhbq?is?2,5-dihydroxy-1,4-benzoquinonato), the larger metallaprism [1](6+) appears to be significantly more stable, and disassembled in the presence of Arg, His and Lys after only 12?h of incubation. Moreover, the reaction with His is not complete, since only 14?% of His reacted after more than 1?week of incubation. Solutions of [1](6+) are also able to catalyse oxidation of the thiol group of Cys and GSH to give the corresponding disulfides and of ascorbic acid to give the corresponding dehydroascorbic acid. However, the results are markedly different from those obtained with metallaprism [2](6+): the oxidation of Cys and ascorbic acid is not complete, and the formation of intermediate adducts could be evidenced. On the other hand, the oxidation of GSH remains fast and is completed after only 12?h. Oxidation of GSH to give the corresponding disulfide may explain its higher in vitro anticancer activity as compared with [2](6+). Our results suggest that metallaprism [1](6+) is more robust than [2](6+), may remain intact in the bloodstream and, therefore, may enter cancer cells undamaged, thus confirming the drug delivery potential for such water-soluble organometallic cages.     

A linear correlation between energy of LMCT band and oxygenation reaction rate of a series of catecholatoiron(III) complexes: initial oxygen binding during intradiol catechol oxygenation

The oxygen reactivity of catecholatoiron(III) complexes has been examined using a series of catecholate ligands as the substrate. All the complexes examined here, [Fe(III)(TPA)(R-Cat)]BPh(4) (1-9) (TPA: tris(pyridin-2-ylmethyl)amine; R-Cat: substituted catecholate ligand, R=3,5-(t)Bu(2) (1), 3,6-(t)Bu2 (2), 3,5-Me2 (3), 3,6-Me2 (4), 4-(t)Bu (5), 4-Me (6), H (7), 4-Cl (8) and 3-Cl (9)), exclusively afforded the intradiol cleaving products of the catecholate ligands upon exposure to O2. It was revealed that 1-7 can be categorized into two classes based on their electrochemical properties; i.e., the complexes having the dialkyl-substituted (group A) and the mono- or non-substituted (group B) catecholate ligands. In spite of their classification, these two groups show a linear correlation between the logarithm of the reaction rate constant with O2 and the energy of the catecholate-to-iron(III) LMCT band, although 2 shows a large negative deviation from the correlation line. Based on this LMCT-energy dependent reactivity of 1 and 3-9 as well as the very low reactivity of 2, we have discussed on the mechanisms of the reaction of [Fe(III)(TPA)(R-Cat)]BPh4 with O2.     

Aluminium(III), chromium(III) and iron(III) complexes with 5-iodouracil and 5-iodouracil-histidine and their antitumour activity

Complexes of the type [Al(HL)(OH)Cl(2)], [M(HL)(OH)(2)Cl] and [M'(HL)(L')(OH)Cl], where HL = 5-iodouracil; HL' = histidine; M = Cr(III), Fe(III) and M' = Al(III), Cr(III), Fe(III), were synthesized and characterized. The complexes are polymeric showing high decomposition points and are insoluble in water and common organic solvents. The mu(eff) values, electronic spectral bands and ESR spectra suggest a polymeric 6-coordinate spin-free octahedral stereochemistry for the Cr(III) and Fe(III) complexes. 5-Iodouracil acts as a monodentate ligand coordinating to the metal ion through the O atom of C((4)) = O while histidine through the O atom of -COO(- ) and the N atom of -NH(2) group. In vivo antitumour effect of 5-iodouracil and its complexes was examined on C(3)H /He mice against P815 murine mastocytoma. As evident from their T/C values, Cr(III) and Fe(III) complexes display significant and higher antitumour activity compared to the 5-iodouracil ligand. The in vitro results of the complexes on the same cells indicate that Cr(III) and Fe(III) complexes show higher inhibition on (3)H-thymidine and (3)H-uridine incorporation in DNA and RNA replication, respectively, at a dose of 5 microg/mL.     

The photorelease of nitrogen monoxide (NO) from pentacyanonitrosyl coordination compounds of group 8 metals.

The photodetachment of NO from [M(II)(CN)5NO]2- with M = Fe, Ru, and Os, upon laser excitation at various wavelengths (355, 420, and 480 nm) was followed by various techniques. The three complexes showed a wavelength-dependent quantum yield of NO production Phi(NO), as measured with an NO-sensitive electrode, the highest values corresponding to the larger photon energies. For the same excitation wavelength the decrease of Phi(NO) at 20 degrees C in the order Fe > Ru > Os, is explained by the increasing M-N bond strength and inertness of the heavier metals. Transient absorption data at 420 nm indicate the formation of the [M(III)(CN)5H2O]2- species in less than ca. 1 micros for M = Fe and Ru. The enthalpy content of [Fe(III)(CN)5H2O]2- with respect to the parent [Fe(II)(CN)5NO]2- state is (190 +/- 20) kJ mol(-1), as measured by laser-induced optoacoustic spectroscopy (LIOAS) upon excitation at 480 nm. The production of [Fe(III)(CN)5H2O]2- is concomitant with an expansion of (8 +/- 3) ml mol(-1) consistent with an expansion of the water bound through hydrogen bonds to the CN ligands plus the difference between NO release into the bulk and water entrance into the first coordination sphere. The activated process, as indicated by the relatively strong temperature dependence of the Phi(NO) values and by the temperature dependence of the appearance of the [Fe(III)(CN)5H2O]2- species, as determined by LIOAS, is attributed to NO detachment in less than ca. 100 ns from the isonitrosyl (ON) ligand (MS1 state).     

Interaction of K7Fe3+P2W17O62H2 with supported bilayer lipid membranes on platinum electrode

The influence of K(7)Fe(3+)P(2)W(17)O(62)H(2) on l-alpha-phosphatidylcholine/cholesterol bilayer lipid membrane on Pt electrode was studied by voltammetry and AC impedance spectroscopy. The interaction of the polyoxometalates with the BLM can promote the access of Ru(NH(3))(6)(3+) and [Fe(CN)(6)](3-/4-) to the electrode surface. It was found that some kind of pores had been formed on the BLM by AFM. The phenomenon is attributed to the interaction of K(7)Fe(3+)P(2)W(17)O(62)H(2) with phosphatidylcholine phosphate groups located in its outer leaflet. Experimental results are helpful to understand the biological activity of the polyoxometalates in vivo.     

Iron(III) complexing ability of carbohydrate derivatives

A solution study on the coordinative ability of galactaric acid (GalAH(2)), d-glucosamine (GlcN) and d-glucosaminic acid (GlcNAH) toward Fe(3+) ion is reported. UV spectroscopic study provides useful information to identify complex species formation and their stability constants are determined by means of potentiometric measurements. GalAH(2) behaves as chelating ligand through carboxylic oxygen and alpha-hydroxylic oxygen in the protonated or dissociated form depending on pH value. Two complex species [Fe(2)GalA(OH)(4)] and Na[FeGalAH(-2)] .2H(2)O are also isolated in the solid state and characterised through IR spectroscopy. GlcNAH also binds the Fe(3+) ion through carboxylic and hydroxylic groups, while NH(2) group is probably involved in metal coordination up to pH 4. GlcN demonstrates low ligating ability at acidic pH and does not prevent metal hydroxyde precipitation.     

O2 reactions at the six-iron active site (H-cluster) in [FeFe]-hydrogenase

Irreversible inhibition by molecular oxygen (O(2)) complicates the use of [FeFe]-hydrogenases (HydA) for biotechnological hydrogen (H(2)) production. Modification by O(2) of the active site six-iron complex denoted as the H-cluster ([4Fe4S]-2Fe(H)) of HydA1 from the green alga Chlamydomonas reinhardtii was characterized by x-ray absorption spectroscopy at the iron K-edge. In a time-resolved approach, HydA1 protein samples were prepared after increasing O(2) exposure periods at 0 °C. A kinetic analysis of changes in their x-ray absorption near edge structure and extended X-ray absorption fine structure spectra revealed three phases of O(2) reactions. The first phase (τ(1) ≤ 4 s) is characterized by the formation of an increased number of Fe-O,C bonds, elongation of the Fe-Fe distance in the binuclear unit (2Fe(H)), and oxidation of one iron ion. The second phase (τ(2) ≈ 15 s) causes a ～50% decrease of the number of ～2.7-? Fe-Fe distances in the [4Fe4S] subcluster and the oxidation of one more iron ion. The final phase (τ(3) ≤ 1000 s) leads to the disappearance of most Fe-Fe and Fe-S interactions and further iron oxidation. These results favor a reaction sequence, which involves 1) oxygenation at 2Fe(H(+)) leading to the formation of a reactive oxygen species-like superoxide (O(2)(-)), followed by 2) H-cluster inactivation and destabilization due to ROS attack on the [4Fe4S] cluster to convert it into an apparent [3Fe4S](+) unit, leading to 3) complete O(2)-induced degradation of the remainders of the H-cluster. This mechanism suggests that blocking of ROS diffusion paths and/or altering the redox potential of the [4Fe4S] cubane by genetic engineering may yield improved O(2) tolerance in [FeFe]-hydrogenase.     

Iron detoxification properties of Escherichia coli bacterioferritin. Attenuation of oxyradical chemistry

Bacterioferritin (EcBFR) of Escherichia coli is an iron-mineralizing hemoprotein composed of 24 identical subunits, each containing a dinuclear metal-binding site known as the "ferroxidase center." The chemistry of Fe(II) binding and oxidation and Fe(III) hydrolysis using H(2)O(2) as oxidant was studied by electrode oximetry, pH-stat, UV-visible spectrophotometry, and electron paramagnetic resonance spin trapping experiments. Absorption spectroscopy data demonstrate the oxidation of two Fe(II) per H(2)O(2) at the ferroxidase center, thus avoiding hydroxyl radical production via Fenton chemistry. The oxidation reaction with H(2)O(2) corresponds to [Fe(II)(2)-P](Z) + H(2)O(2) --> [Fe(III)(2)O-P](Z) + H(2)O, where [Fe(II)(2)-P](Z) represents a diferrous ferroxidase center complex of the protein P with net charge Z and [Fe(III)(2)O-P](Z) a micro-oxo-bridged diferric ferroxidase complex. The mineralization reaction is given by 2Fe(2+) + H(2)O(2) + 2H(2)O --> 2FeOOH((core)) + 4H(+), where two Fe(II) are again oxidized by one H(2)O(2). Hydrogen peroxide is shown to be an intermediate product of dioxygen reduction when O(2) is used as the oxidant in both the ferroxidation and mineralization reactions. Most of the H(2)O(2) produced from O(2) is rapidly consumed in a subsequent ferroxidase reaction with Fe(II) to produce H(2)O. EPR spin trapping experiments show that the presence of EcBFR greatly attenuates the production of hydroxyl radical during Fe(II) oxidation by H(2)O(2), consistent with the ability of the bacterioferritin to facilitate the pairwise oxidation of Fe(II) by H(2)O(2), thus avoiding odd electron reduction products of oxygen and therefore oxidative damage to the protein and cellular components through oxygen radical chemistry.     

Characterization and crystal structure of cadmium(II) halide complexes with amino acids and their derivatives: VII. Crystal structures of aquadibromo(3-aminopropanoic acid)cadmium(II), dichloro(4-aminobutanoic acid)cadmium(II), diaquabis(aminohexanoic acid)cadmium(II) tetrachlorocadmium(II), and dibromo(azetidine-3-carboxylic acid)cadmium(II)

Seven cadmium complexes: [CdX2(Hapro)(H2O)n] (X: Cl(1), Br(2)), [CdX2(Hgaba)] (X: Cl(3), Br(4)), [Cd(Hahex)2(H2O)2][CdCl4] (5), and [CdX2(Haze-3)](H2O)n (X: Cl(6), Br(7)) have been prepared and investigated by means of IR and FT Raman spectra. The crystal and molecular structures of 2, 3, 5 and 7 were determined by a single-crystal X-ray diffraction method. In complex 2, the cadmium atom is in a distorted octahedral geometry, ligated by two carboxyl oxygen atoms of Hapro, a water molecule, and three bromine atoms; one is terminal and each of the other two is bridging two cadmium atoms to make a polymer. The structure of 3 consists of one-dimensional polymers bridged by two chlorine atoms and a carboxyl group. The carboxyl oxygen atoms of Hgaba coordinate forkedly to two cadmium atoms. The cadmium atom of [Cd(Hahex)2(H2O)2]2+ in complex 5 is in a distorted octahedral geometry, ligated by four carboxyl oxygen atoms of two molecules of Hahex and by two water molecules. [Cd(Hahex)2(H2O)2]2+ exists between two layers which are formed of infinite [CdCl4]2- chains. The carboxyl oxygen atoms of Hahex coordinate to the same cadmium atom. In complex 7, the cadmium atom is ligated by two carboxyl oxygen atoms and four bridging bromine atoms to make a polymer.     

Synthesis, structure and spectroscopic properties of two models for the active site of the oxygenated state of cytochrome P450 [corrected

Two dioxygen adducts of thiolato-iron(II) porphyrins, [K(222)][Fe(TPpivP)(SC6HF4)(O2)] 1a and [Na(18c.6)][Fe(TPpivP)(SC6HF4)(O2)] 2 were synthesized by reaction of O2 with five-coordinate, high-spin, cryptated alkali metal thiolato-iron(II) 'picket fence' porphyrinate. They were characterized by visible and infrared spectroscopy: lambda max (log epsilon) = 360 nm (4), 427 nm (4.69), 560 nm (3.69), 610 nm (3.40) for both compounds; v(16O-16O) = 1139 cm-1 in chlorobenzene and fluorobenzene for 1a and 2. Single crystals of composition [K(222)][Fe(TPpivP)(SC6HF4)(O2)].[K(222)](SC6HF4)(C 6H5Cl)(H2O) 1b were obtained by diffusion of pentane/xylene mixtures into chlorobenzene solutions of 1a at -5 degrees C. Single crystals of composition [Na(18c.6)][Fe(TPpivP)(SC6HF4)(O2)] were obtained by slow diffusion of pentane into benzene solutions of 2. Structures of 1b and 2 were studied at 20 degrees C (1b) and -100 degrees C (1b and 2). 1b: space group P2(1)/c (monoclinic), a = 16.806(5) A (1.6806 nm), b = 14.331(4) A (1.4331 nm), c = 52.000(15) A (5.2000 nm), beta = 92.95(2) degrees, V = 12.507 A3 (12.507 nm3), Z = 4, Dcal = 1.28 g.cm-3 (t = 20 degrees C). The final R1 factor was 0.085 for 5238 reflections having I greater than 3 sigma(I). 2: space group P2(1)/c (monoclinic), a = 13.107(3) A (1.3107 nm), b = 27.055(4) A (2.7055 nm), c = 25.029(4) A (2.5029 nm), beta = 96.84(2) degrees, V = 8812 A3 (8.812 nm3), Z = 4, Dcal = 1.18 g.cm-3 (t = -100 degrees C). The final R1 factor was 0.088 for 6587 reflections having I greater than 3 sigma(I). The iron atom is, in both compounds, bonded to the four porphyrinato nitrogens (Np), the sulfur atom of the axial thiolate and one oxygen atom of the axially end-on bonded dioxygen molecule. The average Fe-Np distance found in 1b [1.994(4) A, 0.1994 nm] is not significantly different from that found in 2 [1.993(3) A, 0.1993 nm]. The Fe-S bond length is 2.367(3) A (0.2367 nm) in 1b and 2.365(2) A (0.2365 nm) in 2. The Fe-O1 distances with the oxygen atom of O2 bonded to iron are respectively 1.837(9) A (0.1837 nm) and 1.850(4) A (0.1850 nm). The end-on bonded O2 molecule is disordered in both complexes 1b and 2.(ABSTRACT TRUNCATED AT 400 WORDS)