Self-assembling amphiphilic siderophores from marine bacteria.

Most aerobic bacteria secrete siderophores to facilitate iron acquisition. Two families of siderophores were isolated from strains belonging to two different genera of marine bacteria. The aquachelins, from Halomonas aquamarina strain DS40M3, and the marinobactins, from Marinobacter sp. strains DS40M6 and DS40M8, each contain a unique peptidic head group that coordinates iron(III) and an appendage of one of a series of fatty acid moieties. These siderophores have low critical micelle concentrations (CMCs). In the absence of iron, the marinobactins are present as micelles at concentrations exceeding their CMC; upon addition of iron(III), the micelles undergo a spontaneous phase change to form vesicles. These observations suggest that unique iron acquisition mechanisms may have evolved in marine bacteria.

An affinity label for the anion binding site in ovotransferrin. Implications for iron release to reticulocytes.

Bromopyruvate has been found to be an affinity label for the anion binding site in ovotransferrin. Spectral and radioactive labeling studies indicate that the bromopyruvate first forms a reversible ternary complex with ovotransferrin and iron and then reacts to yield the covalently bound product. This derivative is partially resistant to iron loss by protonation and suggests that protonation and loss of anion is a requisite first step for iron release to reticulocytes.

Fungal ferritins: the ferritin from mycelia of Absidia spinosa is a bacterioferritin.

Two distinct ferritin like iron containing proteins have been identified and isolated from the fungus Absidia spinosa; one from the spores and another from the mycelia. The mycelial protein has been purified and consists of two subunits of approx. 20 kDa. The N-terminal sequences of both subunits have been determined. The holoprotein as isolated contains approx. 750 iron atoms/molecule and exhibits a heme-like UV-Vis spectrum. Based on the heme spectrum and the high degree of sequence homology found, it has been established that the mycelial protein is a bacterioferritin. This is the first example demonstrating the presence of a bacterioferritin in a eukaryotic organism.

Binding of vanadate to human serum transferrin.

Human serum transferrin specifically and reversibly binds 2 equiv of vanadate at the two metal-binding sites of the protein. The vanadium(V)-transferrin complex can be formed either by the addition of vanadate to apotransferrin or by the air oxidation of the vanadyl(IV)-transferrin complex. The formation of the vanadium complex can be blocked by loading the apotransferrin with iron(III), and bound vanadium can be displaced from the protein by the subsequent addition of either gallium(III) or iron(III). The binding constant for the second equiv of vanadate is 10(6.5) in 0.1 M hepes, pH 7.4 at 25 degrees C. The binding constant for the first equiv of vanadate is probably very similar, although no quantitative value could be determined. Although transferrin reacts with the vanadate anion, studies on the transferrin model compound ethylenebis(o-hydroxyphenylglycine) indicate that at pH 9.5, the vanadium is binding at the metal-binding site as a dioxovanadium(V) cation coordinated to two phenolic residues at each binding site. This bound cation appears to be protonated over the pH range 9.5-6.5, as shown by changes in the difference uv spectrum of the transferrin complex, to produce an oxohydroxo species. Further decreases in the pH lead to dissociation of the vanadium-transferrin complex.

Tumor localizing agents: the transport of meso-tetra(p-sulfophenyl) porphine by Vero and HEp-2 cells in vitro.

The mechanism of transport of the tumor localizing agent, meso-tetra(p-sulfophenyl)porphine (TPPS4), was investigated in Vero and HEp-2 cells in vitro. Vero cells proved to be basically impermeable to the porphyrin, but a slow transport was observed. The uptake was linear with time and appeared to be carrier mediated, as it was strongly inhibited by cyanide or low temperature and demonstrated saturation kinetics. Transport in HEp-2 cells was more rapid and non-linear, reaching a plateau after about 2 h. Analysis of this uptake over a 20-fold range of porphyrin concentration revealed it to be biphasic. A low affinity, high capacity component appeared to be unsaturable and was unaffected by low temperature or metabolic inhibitors. This system is probably one of a passive diffusion. The high affinity, low capacity phase is probably carrier mediated. The tumor cells appear to be "leaky" to the porphyrin, with respect to the Vero cells. This may explain part of the localizing ability of TPPS4.

Coordination chemistry of microbial iron transport compounds: rhodotorulic acid and iron uptake in Rhodotorula pilimanae.

The mechanism by which iron uptake is facilitated by the siderophore rhodotorulic acid (RA) in the yeast Rhodotorula pilimanae was investigated with radioactively labeled Fe and RA and kinetically inert, chromic-substituted RA complexes. The weight of the evidence supports a model in which RA mediates iron transport to the cell but does not actually transport iron into the cell. It is proposed that RA exchanges the ferric ion at the cell surface with a membrane-bound chelating agent that completes the active transport of iron into the cell. Uptake of 55Fe in ferric rhodotorulate was much more rapid than uptake of RA itself. Two exchange-inert chromic complexes of RA showed no uptake.

Methylation of (2-methylethanethiol-bis-3,5-dimethylpyrazolyl)methane zinc complexes and coordination of the resulting thioether: relevance to zinc-containing alkyl transfer enzymes.

A series of zinc complexes using a new tripodal, N(2)S, heteroscorpionate ligand (L3SH) that is isostructural and isoelectronic with the well-known N(3) trispyrazolylborates have been methylated in solution and the coordination properties of the resulting thioether examined. This system models the reactivity of zinc-containing enzymes involved in alkyl group transfers such as the DNA repair protein Ada from E. coli, or farnasyl transferase where it has been shown that the thioether resulting from alkyl group transfer remains in the coordination sphere of the zinc. The following complexes have been structurally characterized: [(L3S)ZnI] (1), [(L3SCH(3))ZnI(2)] (2), [(L3SCH(3))ZnI]BF(4) (3), [(L3SCH(3))Zn-mu-bis-acetato-mu-hydroxo-Zn(L3SCH(3))]BF(4) (5), [(L3SCH(3))ZnSPh(F5)]ClO(4) (7), and [(L3SCH(3))(2)Zn](BF(4))(2) (8). Complexes 3, 4, 5, 7, and 8 all display thioether coordination. Thus in the absence of superior anionic ligands, thioethers are reasonably good donors to zinc in either a tetrahedral or octahedral geometry. The methylation of the complex [(L3S)ZnSPh(F5)], which contains two different thiols, produces a single product, 7, where only the aliphatic thiol has been alkylated. This observation validates the suggestion that reactivity in enzymes with multiple zinc-bound thiols could be controlled by differences in thiol pK(a) (Hammes, B. S.; Warthen, C. R.; Crans, D.; Carrano, C. J. J. Biol. Inorg. Chem. 2000, 6, 82. Compound 7 is also of interest in that it resembles the metal ion-binding site of the blue copper protein, azurin.

Affinity labels for the anion-binding site in ovotransferrin.

Bromopyruvate, a known alkylating agent, has previously been reported to function as an affinity label for the anion-binding site in the iron-binding protein ovotransferrin [Patch, M.G., & Carrano, C. J. (1982) Biochim. Biophys. Acta 700, 217-220]. However, the present results indicate that hydroxypyruvate also functions in an almost identical manner, which implies that alkylation of a susceptible nucleophile cannot be the mechanism responsible for the covalent attachment of the anion. Model complexes and amino acid analysis of labeled ovotransferrin suggest that initial Schiff base formation, followed by reduction of the imine bond between the affinity anion and a lysine within the locus of the anion-binding site, accounts for the irreversible labeling. As expected, the covalently attached anions render the iron in the ovotransferrin-iron-anion ternary complex much more resistant to loss at low pH. It is proposed that the covalently labeled protein be used to test the hypothesis that iron removal from transferrin occurs by protonation and loss of the anion in low-pH lysosomal vesicles.

Identification of the ferrioxamine B receptor, FoxB, in Escherichia coli K12.

The photoreactive p-azidobenzoyl analog of ferrioxamine B was used to show that ferrioxamine-B-mediated iron transport is separate and distinct from coprogen-mediated iron transport in Escherichia coli. Photolysis of this analog inhibited uptake of [59Fe]ferrioxamine B but not [59Fe]ferrichrome. Conversely, photolysis of the p-azidobenzoyl analog of coprogen B inhibited uptake of [59Fe]coprogen but not [59Fe]ferrioxamine B or [59Fe]ferrichrome. Photolabeling of outer membranes with p-azidobenzoyl-[59Fe]ferrioxamine B resulted in the labeling of two iron-regulated peptides with molecular masses of about 66 and 26 kDa. Expression of these peptides was increased when ferrioxamine B was the sole iron source. Both peptides were present in outer membrane preparations of the fhuF mutant H1717, but the 66 kDa peptide was not inducible. These results are evidence for an outer membrane receptor in E. coli unique for linear ferrioxamines.

Localization of meso-tetra(p-sulfophenyl)porphine in murine sarcoma virus-induced tumor-bearing mice.

meso-Tetra(p-sulfophenyl)porphine (TPPS4) has been found to accumulate in the tumors of mice bearing a murine sarcoma virus-induced rhabdomyosarcoma to a greater degree than in all other tissues except for the kidney. Tumor to tissue ratios of from 3:1 (tumor to liver) to 9:1 (tumor to muscle) were observed. The uptake of TPPS4 was found to be dose dependent, and the highest tumor to tissue ratios were found 96 hours after injection. The water-soluble TPPS4 appears to be cleared through the kidneys and absolute concentrations there are reduced with increasing time after injection. These data are compared with previous studies on TPPS4 localization in Walker 256 carcinosarcoma-bearing mice. In view of this localizing ability and its relative lack of toxicity, TPPS4 seems to represent an ideal candidate for a tumor-localizing agent if high levels of the porphyrin in the kidneys can be reversed.

Intracellular localization of meso-Tetra(p-sulfophenyl)porphine: a potential tumor localizing agent.

The mechanism of meso-tetra (p-sulfophenyl)porphine (TPPS4) localization was examined in Vero and HEp-2 cells in vitro. TPPS4 was found to be relatively nontoxic to both cell types at concentrations up to 200 microgram/ml. However, changes in cellular ultrastructure were observed by electron microscopy, consisting mainly of an increase in membrane-bound vacuoles. The nature of the new structures has not been determined. Both fluorescence microscopy and cellular fractionation indicate that TPPS4 is localized inside the cells studied. Considerable differences in the pattern of localization between the two cell types were found. The TPPS4 in the Vero cells was found to be mainly in the soluble fraction and the red fluorescence was found to be diffuse throughout the cytoplasm. The HEp-2 cells showed TPPS4 fluorescence associated with various cell organelles, predominatly the nucleolus. [14C]-TPPS4 was found in many cellular components but predominantly it was in the soluble and protein fractions. These differences may be significant in the understanding of in vivo localization of this porphyrin in neoplastic tissue.

Methylation of neutral pseudotetrahedral zinc thiolate complexes: model reactions for alkyl group transfer to sulfur by zinc-containing enzymes.

Eight scorpionate-zinc thiolate complexes, [(L1O)ZnSPh], [(L1O)ZnSPhF5], [(L1O)ZnSBz], [(L1O)ZnSPh2,6-Me], [(L1O)ZnSPh2,4-Me], [(L1O)ZnSPh4-NO2], [(TpPh,Ph)ZnSPh], and [(L2S)ZnSPh], were reacted with methyl iodide in chloroform, liberating the corresponding methyl thioethers as determined by 1H NMR. Three of these complexes are new and their synthesis and structural characterization are reported here. Weak alkylating agents such as trimethyl phosphate failed to undergo methyl transfer to the zinc thiolates under these conditions. Analysis of kinetic data as a function of concentration, temperature, pKa of the exogenous thiolate, and donor atom of the tripodal ligand are consistent with a mechanism where the zinc-bound thiolate is the active nucleophile in an associative-type methyl transfer reaction. Our model studies also provide experimental evidence to support the hypothesis that some enzymes can use the charge of the metal coordination site to modulate catalytic activity.

Interaction of oxo-bridged vanadium(III) phenanthroline and bipyridine dimers with DNA.

Cationic mu-oxo V(III) dimers of the type [V2OL4Cl2]2+ (L = 1,10-phenanthroline, 3,4,7,8-tetramethyl-1,10-phenanthroline, 4,7-diphenyl-1,10-phenanthroline; or 2,2'-bipyridine) are shown to interact very strongly with DNA and to lead ultimately to its degradation. Spectroscopic binding studies, electrophoreses, DNA melting temperature experiments, and other tests on the parent 1,10-phenthroline complex all yield results consistent with tight binding. However, the exact nature of the binding--i.e., intercalative, groove binding, electrostatic, or covalent--remains unclear. Resonance Raman spectroscopy is found to be a powerful method for studying the interaction of these mu-oxo V(III) dimers with DNA and shows that in frozen aqueous solution, the parent complex [V2O(phen)4Cl2]2+ undergoes initial aquation, followed by the reaction of the aquated species with the DNA. Once the V(III) dimer is bound to the DNA, redox takes place, leading to the formation of alkaline-sensitive lesions. Hydrogen peroxide is implicated as a partner in this redox event, based on the effects of the enzymes SOD and catalase.