Oxygen and nitric oxide affinities of iron(II) ‘capped’ porphyrins

Oxygen and NO binding constants are reported for Fe(Por)(B) [1] complexes. The results show a good correlation between O₂ and NO affinities, and this is attributed to the similar bent structures of the FeOO and FeNO moieties. Evidence is presented to show that iron(II) C₃-capped porphyrins have a low affinity for O₂ because of ligand coordination on the cavity side of the cap.     

Poly(ethylene glycol)-conjugated human serum albumin including iron porphyrins: surface modification improves the O2-transporting ability

Artificial O2-carrying hemoprotein composed of human serum albumin including tetrakis(o-amidophenyl)porphinatoiron(II) (Fe4P or Fe3P) [HSA-FeXP] has been modified by maleimide- or succinimide-terminated poly(ethylene glycol) (PEG), and the formed PEG bioconjugates have been physicochemically characterized. 2-Iminothiolane (IMT) reacted with the amino groups of Lys to create active thiol groups, which bind to alpha-maleimide-omega-methoxy PEG [Mw: 2-kDa (PEG(M2)), 5-kDa (PEG(M5))]. On the other hand, alpha-succinimidyl-omega-methoxy PEG [Mw: 2-kDa (PEG(S2)), 5-kDa (PEG(S5))] directly binds to Lys residues. MALDI-TOF MS of the PEG-conjugated HSA-FeXP showed distinct molecular ion peaks, which provide an accurate number of the PEG chains. In the case of PEG(MY)(HSA-FeXP), the spectroscopic assay of the thiol groups also provided the mean of the binding numbers of the polymers, and the degree of the modification was controlled by the ratio of [IMT]/[HSA]. The viscosity and colloid osmotic pressures of the 2-kDa PEG conjugates (phosphate-buffered saline solution, [HSA] = 5 g dL(-1)) were almost the same as that of the nonmodified one, whereas the 5-kDa PEG binding increased the rheological parameters. The presence of flexible polymers on the HSA surface retarded the association reaction of O2 to FeXP and stabilized the oxygenated complex. Furthermore, PEG(MY)(HSA-FeXP) exhibited a long circulation lifetime of FeXP in rats (13-16 h). On the basis of these results, it can be concluded that the surface modification of HSA-FeXP by PEG has improved its comprehensive O2-transporting ability. In particular the PEG(MY)(HSA-FeXP) solution could be a promising material for entirely synthetic O2-carrying plasma expander as a red cell substitute.     

Human serum albumin bearing covalently attached iron(II) porphyrins as O2-coordination sites

Tetrakis{(alpha,alpha,alpha,alpha-o-pivalamido)phenyl}porphinatoiron(II) with a bifunctional tail possessing an axially coordinated imidazolyl group and a protein attachable succinimidyl(glutamyl) group (FeP-GluSu) has been synthesized. It can efficiently react with the lysine residues of recombinant human serum albumin (rHSA), giving a new albumin-heme conjugate [rHSA(FeP-Glu)]. MALDI-TOFMS showed a distinct molecular ion peak at m/z 70 643, which indicates that three FeP-Glu molecules were covalently linked to the rHSA scaffold. The binding number of FeP-Glu is approximately three (mol/mol) and independent of the mixing ratio. The CD spectrum and Native PAGE revealed that the albumin structure remained unaltered after the covalent bonding of the hemes. This rHSA(FeP-Glu) conjugate can bind and release O2 reversibly under physiological conditions (pH 7.3, 37 degrees C) in the same manner as hemoglobin and myoglobin. The O2-adduct complex had a remarkably long lifetime (tau(1/2): 5 h). The O2-binding affinity [P(1/2)O2: 27 Torr] was identical to that of human red cells. Laser flash photolysis experiments gave the O2- and CO-association rate constants and suggested that there are two different geometries of the imidazole binding to the central ion.     

Altered molecular form of acyl carrier protein associated with beta-ketoacyl-acyl carrier protein synthase II (fabF) mutants.

Acyl carrier protein (ACP) is a required cofactor for fatty acid synthesis in Escherichia coli. Mutants lacking beta-ketoacyl-ACP synthase II activity (fabF1 or fabF3) possessed a different molecular species of ACP (F-ACP) that was separated from the normal form of the protein by conformationally sensitive gel electrophoresis. Synthase I mutants contained the normal protein. Complementation of fabF1 mutants with an F' factor harboring the wild-type synthase II allele resulted in the appearance of normal ACP, whereas complementation with an F' possessing the fabF2 allele (a mutation that produces a synthase II enzyme with altered catalytic activity) resulted in the production of both forms of ACP. The structural difference between F-ACP and ACP persisted after the removal of the 4'-phosphopantetheine prosthetic group, and both forms of the protein had identical properties in an in vitro fatty acid synthase assay. Both ACP and F-ACP were purified to homogeneity, and their primary amino acid sequences were determined. The two ACP species were identical but differed from the sequence reported for E. coli E-15 ACP in that an Asn instead of an Asp was at position 24 and an Ile instead of a Val was at position 43. Therefore, F-ACP appears to be a modification of ACP that is detected when beta-ketoacyl-ACP synthase II activity is impaired.     

Ligand and carbon monoxide affinities of iron(II) ‘C4-capped’ porphyrins

Ligand and CO binding constants are reported for Fe(C₄-Cap) [1]. The results show that the CO affinities are Fe(C₂-Cap)(B) ?Fe(C₃-Cap)(B) > Fe(C₄-Cap)(B). Also the CO affinities are Fe(Cap)(1,5-DCIm) > Fe(Cap) (1,2-Me₂Im). These results are explained in terms of the possible steric factors involved.     

Synthesis of an iron(II)-braced proline-II tripod protein

Summary This paper describes the engineering of braced tripod proteins for use as molecular frameworks. Specifically, a 30-residue tripod-shaped protein with three proline-II helical legs braced by an iron(II)tris(bipyridine) complex was modularly designed, chemically synthesized, and biophysically characterized. Three copies of a 10-residue leg peptide were covalently linked through sulfide bonds to an N-terminal apex (1,3,5-tris(methylene)benzene) and by amide bonds to the brace (Fe^II (Mbc)₃: Mbc is 4′-methyl-2,2′-bipyridine-4-carbonyl). The leg peptide (H-Cys-Pro₅-Pra(Mbc)-Pro₃-NH₂: Pra iscis-4-amino-l-proline) was assembled by the solid-phase method using Boc-Pra(Mbc)-OH, which was synthesized in 75% overall yield by coupling Mbc-OH to the 4-amino group of Boc-Pra-OCH₃ and saponifying the methyl ester group. The iron(II)-braced tripod was assembled by S-alkylation of three copies of the leg peptide with 1,3,5-tris(bromomethyl)benzene followed by ligation of Fe²⁺ to the resulting unbraced tripod. The CD spectrum of the iron(II)-braced tripod showed a positive MLCT band at 570 nm and a negative π-π^* band at 312 nm, so its Fe^II(Mbc)₃ brace was predominantly in the Δ configuration. In a mostly acetonitrile solution at 25°C, the leg peptide and the unbraced tripod isomerized from the proline-II helical form into the proline-I helical form but the iron(II)-braced tripod remained in the proline-II helical form.     

Synthesis of an iron(II)-braced proline-II tripod protein

This paper describes the engineering of braced tripod proteins for use as molecular frameworks. Specifically, a 30-residue tripod-shaped protein with three proline-II helical legs braced by an iron(II)tris(bipyridine) complex was modularly designed, chemically synthesized, and biophysically characterized. Three copies of a 10-residue leg peptide were covalently linked through sulfide bonds to an N-terminal apex (1,3,5-tris(methylene)benzene) and by amide bonds to the brace (FeII(Mbc)3: Mbc is 4-methyl-2,2-bipyridine-4-carbonyl). The leg peptide (H-Cys-Pro5-Pra(Mbc)-Pro3-NH2: Pra is cis-4-amino-l-proline) was assembled by the solid-phase method using Boc-Pra(Mbc)-OH, which was synthesized in 75% overall yield by coupling Mbc-OH to the 4-amino group of Boc-Pra-OCH3 and saponifying the methyl ester group.The iron(II)-braced tripod was assembled by S-alkylation of three copies of the leg peptide with 1,3,5-tris(bromomethyl)benzene followed by ligation of Fe2+ to the resulting unbraced tripod. The CD spectrum of the iron(II)-braced tripod showed a positive MLCT band at 570 nm and a negative –* band at 312 nm, so its FeII(Mbc)3 brace was predominantly in the configuration. In a mostly acetonitrile solution at 25 °C, the leg peptide and the unbraced tripod isomerized from the proline-II helical form into the proline-I helical form but the iron(II)-braced tripod remained in the proline-II helical form.     

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.     

Acyl carrier protein (ACP) inhibition and other differences between beta-ketoacyl synthase (KAS) I and II

Escherichia coli beta-ketoacyl synthases (KAS) I and II carry out the elongation steps in fatty acid synthesis. Analyses using the cross-linker BS(3) [bis(sulphosuccinimidyl) suberate] and surface-enhanced laser desorption/ionization-time-of-flight MS disclosed only monomeric and dimeric forms of KAS II, whereas KAS I also forms higher multimers. The binding affinities for KAS I and KAS II to C(14)-acyl carrier protein (ACP) as well as for C(14)-ACP to KAS I and KAS II were determined. KAS I is sensitive to the ACP released during the transfer reaction, with 50% inhibition at 0.17 microM ACP close to the physiological concentration of ACP (0.13 microM). KAS I and II also differ in carrying out the decarboxylation step of the elongation reaction.     

Interaction of Nickel(II) with histones: in vitro binding of nickel(II) to the core histone tetramer

The absorption spectra of Ni(II) bound to the core histone tetramer, (H3-H4)2, of chicken erythrocytes in 500 mM NaCl + 100 mM phosphate (pH 7.4) were recorded. A charge transfer band was seen at 317 nm, characteristic of a bond between Ni(II) and the sulfur atom of Cys-110 of histone H3. The conditional affinity constants for Ni(II) binding at pH 7.4 for low and high Ni(II) saturation (log Kc = 4.26 +/- 0.02 and 5.26 +/- 0.11 M-1, respectively) were calculated from spectrophotometric titrations with the use of this band. The binding of Ni(II) to (H3-H4)2 is proposed to involve the Cys-110 and His-113 of different H3 molecules within the tetramer. The competition between histones and low-molecular-weight chelators for Ni(II) in the cell nucleus, histidine and glutathione, is discussed on the basis of the above results, indicating that histone H3 is very likely to bind Ni(II) dissolved intracellularly from phagocytosed particulate nickel compounds.     

Photoinduced reduction of water by tin(IV) and ruthenium(II) porphyrins

In the paper are presented new photoredox systems for the reduction of water in which water- soluble Sn(IV) and Ru(II) porphyrins have been used as photosensitizers It has been found that during the photolysis of water Sn(IV) porphyrin underwent photoreduction whereas Ru(II) porphyrin underwent photooxidation. The successive photo- products of Sn(IV) porphyrin in the reaction from EDTA were, first, Sn(IV) chlorin and, second, Sn(IV) bacteriochlorin. In the experiments on the photo- generation of hydrogen, a correlation between the rates of hydrogen evolution and the reduction potentials of the electron carriers has been observed. The highest rate of hydrogen generation by means of Sn(IV) and Ru(II) porphyrins has been found for those electron carriers whose values of reduction potentials were tau; 0.55 and tau; 0.45 V. In the case of Ru(II) porphyrin, the rate of hydrogen evolution additionally depended on the molecular structure of the electron carrier. It has been found that during the water photolysis, viologens show a tendency to form their respective complexes with Ru(II) porphyrin, but only when they occur in a one-electron reduced form in the solution.     

Toxic effects of mercury(II), cadmium(II) and lead(II) porphyrins on Trypanosoma brucei brucei growth

The effects of free mercury(II), cadmium(II) and lead(II) ions and their metalloporphyrin-derivatives on Trypanosoma brucei brucei growth in culture were studied. All experiments were conducted in the dark. IC(50) values on growth obtained in 24-h time-course experiments were 1.5 x 10(-7), 2.4 x 10(-6), 4.4 x 10(-6) and 2.6 x 10(-5) M for mercury(II) porphyrin, cadmium(II) porphyrin, lead(II) porphyrin and free base porphyrin, respectively. While the IC50 values for Hg2+, Cd2+ and Pb2+ were 3.6 x 10(-6), 1.5 x 10(-5) and 1.6 x 10(-5) M, respectively. These results clearly indicate that the toxicity of the metalloporphyrin complexes of mercury(II), cadmium(II) and lead(II) to T. b. brucei parasites was much higher compared to their free metal ions and free base porphyrin at low concentrations. It was also observed after 8 h incubation that the metalloporphyrins were effective in inhibiting the division of the parasites at concentrations >1.25 x 10(-7) M for mercury(II) porphyrin, concentrations >1.2 x 10(-6) M for cadmium(II) and lead(II) porphyrins and at concentrations >3.6 x 10(-6) M for Hg2+ ion. These observations were not detected in samples treated with the free metal ions and the free base porphyrin at the same concentrations. Interestingly, trypanosomes treated with metalloporphyrin complexes displayed different morphological features from those cells treated with free base porphyrin or metal ions. The chemotherapeutic potential of the metalloporphyrins of H2TMPyP for treatment of African trypanosomiasis is discussed.     

Amidino-complexes of iron(II) and (III)

Lithioamidines {R′N(Li)C(R)NR′, I; R = CH₃, R′ = C₆H₅, p-CH₃,C₆H₄} react with iron(III) chloride

in monoglyme to produce navy-blue, high spin Fe{R′NC(R)NR′}₃ complexes which are extremely air and moisture sensitive. The corresponding reaction when R = R′ = C₆H₅ produces a soluble red complex and an air-stable green complex, whereas when R = H, R′ = C₆H₅ and R = R′ = C₆H₅ and the reaction is started at ca. ?20°, red and green complexes respectively are formed. Though all the complexes are formulated Fe{R′NC(R)NR′}₃, their properties reflect association through bridging amidino-groups. Iron(II) chloride reacts with I(R = CH₃, R′ = p-CH₃C₆H₄) to form two complexes, one crimson and soluble in organic solvents, and one brown and insoluble, which are fomulated [Fe{R′NC(R)NR′}₂]_n. The iron(III) complexes failed to react with, or were decomposed by, a variety of reducing, electrophilic and nucleophilic reagents, though blue Fe{p-CH₃C₆H₄NC(CH₃)N-p-CH₃C₆H₄}₃ reacts readily with nitric oxide to form a purple addition complex from which the N-nitroso-compound p-CH₃C₆H₄NC(CH₃)N(NO)-p-CH₃C₆H₄ was obtained in high yield. Treatment of the corresponding brown iron(II) complex with nitric oxide gave no reaction.     

Complexes of chromium(II) and iron(II) with hexamethylphosphoramide

The chromium(II) complexes CrX₂(HMPA)₂, in which X = Cl or Br and HMPA is hexamethylphosphoramide, and Cr(HMPA)₄(BF₄)₂ have been prepared. The effective magnetic moments show little deviation from the value expected for high spin chromium(II) from room temperature to liquid nitrogen temperature. The diffuse reflectance spectra suggest that the chromium ions are in a strongly distorted six coordinate environment. The iron(II) complexes, FeX₂(HMPA)₂, X = Br or I, and [Fe(HMPA)_4](BF₄)₂, from their magnetic behaviour and Mössbauer and electronic spectra, contain tetrahedral iron(II). The isomer shift of the last complex is the most positive so far reported for a tetrahedral iron(II) complex.     

Key residues responsible for acyl carrier protein and beta-ketoacyl-acyl carrier protein reductase (FabG) interaction

Fatty acid synthesis in bacteria is catalyzed by a set of individual enzymes collectively known as type II fatty-acid synthase. Each enzyme interacts with acyl carrier protein (ACP), which shuttles the pathway intermediates between the proteins. The type II enzymes do not possess primary sequence similarity that defines a common ACP-binding site, but rather are hypothesized to possess an electropositive/hydrophobic surface feature that interacts with the electronegative/hydrophobic residues along helix alpha2 of ACP (Zhang, Y.-M., Marrakchi, H., White, S. W., and Rock, C. O. (2003) J. Lipid Res. 44, 1-10). We tested this hypothesis by mutating two surface residues, Arg-129 and Arg-172, located in a hydrophobic patch adjacent to the active site entrance on beta-ketoacyl-ACP reductase (FabG). Enzymatic analysis showed that the mutant enzymes were compromised in their ability to utilize ACP thioester substrates but were fully active in assays with a substrate analog. Direct binding assays and competitive inhibition experiments showed that the FabG mutant proteins had reduced affinities for ACP. Chemical shift perturbation protein NMR experiments showed that FabG-ACP interactions occurred along the length of ACP helix alpha2 and extended into the adjacent loop-2 region to involve Ile-54. These data confirm a role for the highly conserved electronegative/hydrophobic residues along ACP helix alpha2 in recognizing a constellation of Arg residues embedded in a hydrophobic patch on the surface of its partner enzymes, and reveal a role for the loop-2 region in the conformational change associated with ACP binding. The specific FabG-ACP interactions involve the most conserved ACP residues, which accounts for the ability of ACPs and the type II proteins from different species to function interchangeably.     

Angular-overlap analysis of the iron(II) site in [2Fe-2S] clusters

The angular-overlap model for Fe(II)S4 centres is used to obtain structural information form the experimental data available for the gav approximately equal to 1.96 and gav approximately equal to 1.91 classes of [2Fe-2S] ferredoxin, showing that it is possible to translate the parameters obtained by Bertrand and Gayda in their non-additive ligand field model (Bertrand, P. and Gayda J.P. (1979 and 1980) Biochim. Biophys. Acta 579, 107-121 and 625, 337-342, respectively) into an additive one. The analysis of the e lambda (lambda = sigma or pi) parameters allowed us to conclude that the Fe(II)S4 chromophores of the two types of [2Fe-2S] metallo-protein are similar to each other, being possible to reproduce nicely the different g tensors introducing only small variations in the angular and bonding parameters.     

Trispyrazolylmethane piano stool complexes of iron(II) and cobalt(II)

A series of cationic trispyrazolylmethane complexes of the general form [Tm^RM(CH₃CN)₃]²⁺ (Tm = tris(pyrazolyl)methane, 1, R = 3,5-Me₂, M = Fe(II); 2, R = 3-Ph, M = Fe(II); 3, R = 3,5-Me₂, M = Co(II); 4, R = 3-Ph, M = Co(II)) with ‘piano-stool’ structures was prepared by the reaction of the N₃tripodal ligands (Tm^R)with [(CH₃CN)₆M](BF₄)₂ in a 1:1 stoichiometric ratio. Magnetic susceptibility measurements indicate that all four complexes with BF₄⁻ counter anions are paramagnetic, high-spin systems in the solid state with μ_eff at high temperatures of 5.2 (1, S = 2), 5.4 (2, S = 2), 4.9 (3, S = 3/2) and 4.6 (4, S = 3/2) BM, respectively. Comparisons of bond lengths from the metal centre to the Tm^R nitrogen donors, and from the metal centre to the acetonitrile nitrogen donors indicate that the neutral tripodal ligands appear to be more weakly coordinated to the metal centre than are the acetonitrile ligands. Reactions of these tripodal complexes with bidentate phosphine ligands, such as 1,2-diphosphinoethane or 1,2-bis(diallylphosphino)ethane leads to displacement of the tripodal ligand, or to the formation of more thermally stable bis-ligand complexes M(Tm^R)₂ (R = 3,5-dimethyl).     

X-ray crystal structure of Mycobacterium tuberculosis beta-ketoacyl acyl carrier protein synthase II (mtKasB)

Mycolic acids are long chain alpha-alkyl branched, beta-hydroxy fatty acids that represent a characteristic component of the Mycobacterium tuberculosis cell wall. Through their covalent attachment to peptidoglycan via an arabinogalactan polysaccharide, they provide the basis for an essential outer envelope membrane. Mycobacteria possess two fatty acid synthases (FAS); FAS-I carries out de novo synthesis of fatty acids while FAS-II is considered to elongate medium chain length fatty acyl primers to provide long chain (C(56)) precursors of mycolic acids. Here we report the crystal structure of Mycobacterium tuberculosis beta-ketoacyl acyl carrier protein synthase (ACP) II mtKasB, a mycobacterial elongation condensing enzyme involved in FAS-II. This enzyme, along with the M. tuberculosis beta-ketoacyl ACP synthase I mtKasA, catalyzes the Claisen-type condensation reaction responsible for fatty acyl elongation in FAS-II and are potential targets for development of novel anti-tubercular drugs. The crystal structure refined to 2.4 A resolution revealed that, like other KAS-II enzymes, mtKasB adopts a thiolase fold but contains unique structural features in the capping region that may be crucial to its preference for longer fatty acyl chains than its counterparts from other bacteria. Modeling of mtKasA using the mtKasB structure as a template predicts the overall structures to be almost identical, but a larger entrance to the active site tunnel is envisaged that might contribute to the greater sensitivity of mtKasA to the inhibitor thiolactomycin (TLM). Modeling of TLM binding in mtKasB shows that the drug fits the active site poorly and results of enzyme inhibition assays using TLM analogues are wholly consistent with our structural observations. Consequently, the structure described here further highlights the potential of TLM as an anti-tubercular lead compound and will aid further exploration of the TLM scaffold towards the design of novel compounds, which inhibit mycobacterial KAS enzymes more effectively.