CuI and H2O2 Inactivate and FeII Inhibits [Fe]‐Hydrogenase at Very Low Concentrations

[Fe]‐Hydrogenase (Hmd) catalyzes reversible hydride transfer from H₂. It harbors an iron‐guanylylpyridinol as a cofactor with an Fe^II that is ligated to one thiolate, two COs, one acyl‐C, one pyridinol‐N, and solvent. Here, we report that Cu^I and H₂O₂ inactivate Hmd (half‐maximal rates at 1 μM Cu^I and 20 μM H₂O₂) and that Fe^II inhibits the enzyme with very high affinity (K_i=40 nM ). Infrared and EPR studies together with competitive inhibition studies with isocyanide indicated that Cu^I exerts its inhibitory effect most probably by binding to the active site iron‐thiolate ligand. Using the same methods, it was found that H₂O₂ binds to the active‐site iron at the solvent‐binding site and oxidizes Fe^II to Fe^III. Also it was shown that Fe^II reversibly binds away from the active site iron, with binding being competitive to the organic hydride acceptor; this inhibition is specific for Fe^II and is reminiscent of that for the [FeFe]‐hydrogenase second iron, which specifically interacts with H₂.     

Artificial Maturation of the Highly Active Heterodimeric [FeFe] Hydrogenase from Desulfovibrio desulfuricans ATCC 7757

Hydrogenases catalyze the reduction of protons and oxidation of molecular hydrogen with high turnover frequencies and low overpotentials under ambient conditions. The heterodimeric [FeFe] hydrogenase from Desulfovibrio desulfuricans has an exceptionally high activity, and can be purified aerobically in an oxygen-stable inactive state. Recently, it was demonstrated that monomeric [FeFe] hydrogenases produced recombinantly in Escherichia coli can be artificially maturated by simply incubating the inactive “apo” enzymes with the synthetic [2Fe] cofactor mimic [Fe₂(adt)(CO)₄(CN)₂]²⁻. Here, we use the same technique to produce the heterodimeric “apo” hydrogenase from D. desulfuricans in E. coli with a high yield and purity, and maturate the “apo” enzyme with [Fe₂(adt)(CO)₄(CN)₂]²⁻ to generate fully active “holo” enzyme. Interestingly, the rate of the artificial maturation process with D. desulfuricans is significantly slower than that for all other hydrogenases tested so far. The artificially maturated enzyme is spectroscopically and electrochemically identical to the native enzyme and shows high rates of hydrogen production (3700 s⁻¹) and hydrogen oxidation (63,000 s⁻¹). We expect that our highly efficient production method will facilitate future studies of this enzyme and other related [FeFe] hydrogenases from Desulfovibrio species.     

The [4Fe-4S]-Cluster of HydF is not Required for the Binding and Transfer of the Diiron Site of [FeFe]-Hydrogenases

The active site of [FeFe]-hydrogenases contains a cubane [4Fe-4S]-cluster and a unique diiron cluster with biologically unusual CO and CN⁻ ligands. The biogenesis of this diiron site, termed [2Fe_H], requires the maturation proteins HydE, HydF and HydG. During the maturation process HydF serves as a scaffold protein for the final assembly steps and the subsequent transfer of the [2Fe_H] precursor, termed [2Fe_P], to the [FeFe]-hydrogenase. The binding site of [2Fe_P] in HydF has not been elucidated, however, the [4Fe-4S]-cluster of HydF was considered as a possible binding partner of [2Fe_P]. By targeting individual amino acids in HydF from Thermosipho melanesiensis using site directed mutagenesis, we examined the postulated binding mechanism as well as the importance and putative involvement of the [4Fe-4S]-cluster for binding and transferring [2Fe_P]. Surprisingly, our results suggest that binding or transfer of [2Fe_P] does not involve the proposed binding mechanism or the presence of a [4Fe-4S]-cluster at all.     

Synthesis and Properties of Conducting Bridged Macrocyclic Metal Complexes

Stacked bridged macrocyclic metal complexes of the type shown in Fig. 1A, with pyrazine and 1,4-diisocyanobenzene as the bridging ligands L, phthalocyanine (Pc) and tetrabenzoporphyrine (TBP) as the macrocycle (Mac), and Fe and Ru as the central metal atoms M, are doped with iodine. The properties and conductivities of the doped compounds [MacML]_n are reported. The synthesis and properties of type-B polymers (Fig. 1B) ([PcMCN]_n, M = Co, Rh, Fe, Mn, Cr) with CN as the bridging ligand are described. With Co and Fe these polymers show room temperature conductivities around 10⁻¹ S/cm without doping, which are in the same range as the iodine doped [PcML]_n-compounds. [PcCo(SCN)]_n and [TBPCoCN]_n are additional examples of type B polymers.     

Application of a Synthetic Ferredoxin-Inspired [4Fe4S]-Peptide Maquette as the Redox Partner for an [FeFe]-Hydrogenase

‘Bacterial-type’ ferredoxins host a cubane [4Fe4S]^2+/+ cluster that enables these proteins to mediate electron transfer and facilitate a broad range of biological processes. Peptide maquettes based on the conserved cluster-forming motif have previously been reported and used to model the ferredoxins. Herein we explore the integration of a [4Fe4S]-peptide maquette into a H₂-powered electron transport chain. While routinely formed under anaerobic conditions, we illustrate by electron paramagnetic resonance (EPR) analysis that these maquettes can be reconstituted under aerobic conditions by using photoactivated NADH to reduce the cluster at 240 K. Attempts to tune the redox properties of the iron-sulfur cluster by introducing an Fe-coordinating selenocysteine residue were also explored. To demonstrate the integration of these artificial metalloproteins into a semi-synthetic electron transport chain, we utilize a ferredoxin-inspired [4Fe4S]-peptide maquette as the redox partner in the hydrogenase-mediated oxidation of H₂.     

Silicon core–iron siloxane shell nanoparticle polymer composites with multiferroic properties

Multiferroic (MF) composites based on nanoparticles consisting of a silica core and a shell of spin-variable Fe(III) complexes in a polymer matrix (polystyrene) were synthesized and characterized by different methods. The nanoparticles had the formula 80SiO₂·20{Fe[OSi(Me)(OEt)₂]₃}, and their particle size was on the order of 5–7 nm. Dielectric and electron spin resonance studies showed the presence of two types of Fe ions in the nanocomposite. Iron ions in the low-spin state [Fe(III)-LS] and iron ions in the high-spin state [Fe(III)-HS], which were bound by indirect exchange interactions through oxygen and silicon atoms {[Fe(III)-LS]─O─Si─O─[Fe(III)-HS]} were responsible for the MF properties of the composites with core–shell nanoparticles. © 2019 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2019 , 136, 47681.     

Electron Paramagnetic Resonance and Magnetic Circular Dichroism Spectra of the Nitrogenase M Cluster Precursor Suggest Sulfur Migration upon Oxidation: A Proposal for Substrate and Inhibitor Binding

The active site of the nitrogen-fixing enzyme Mo-nitrogenase is the M cluster ([MoFe₇S₉C ⋅ R-homocitrate]), also known as the FeMo cofactor or FeMoco. The biosynthesis of this highly complex metallocluster involves a series of proteins. Among them, NifB, a radical-SAM enzyme, is instrumental in the assembly of the L cluster ([Fe₈S₉C]), a precursor and all-iron core of the M cluster. In the absence of sulfite, NifB assembles a precursor form of the L cluster called the L* cluster ([Fe₈S₈C]), which lacks the final ninth sulfur. EPR and MCD spectroscopies are used to probe the electronic structures of the paramagnetic, oxidized forms of both the L and L* clusters, labeled L^Ox and [ L* ] ^Ox . This study shows that both L^Ox and [ L* ] ^Ox have nearly identical EPR and MCD spectra, thus suggesting that the two clusters have identical structures upon oxidation; in other words, a sulfur migrates away from L^Ox following oxidation, thereby rendering the cluster identical to [ L* ] ^Ox . It is proposed that a similar migration could occur to the M cluster upon oxidation, and that this is an instrumental part of both M cluster formation and nitrogenase substrate/inhibitor binding.     

NO Adsorption on Ex-Framework [Fe,X]MFI Catalysts: Novel IR Bands and Evaluation of Assignments

IR spectra of NO adsorbed on isomorphously substituted [Fe,Al]MFI, [Fe,Ga]MFI and [Fe]MFI after steaming at 873 K in 30 vol% H₂O are presented. On ex-[Fe,Al]MFI, NO adsorption leads to bands at 2133 cm^-1 and a doublet at 1886 and 1874 cm^-1. The 2133 cm^-1 band is assigned to NO⁺ occupying cationic positions in the zeolite structure. Of the doublet, the 1874 cm^-1 band is much more susceptible to reaction with O₂ than the 1886 cm^-1 band, yielding adsorbed NO₂ with an absorption frequency of 1635 cm^-1. After evaluation of the constitution of the catalyst and (sometimes contradictory) literature assignments, the 1886 cm^-1 band is assigned to NO adsorbed on Fe ions located in isolated positions, and/or (FeO)_n clusters inside the zeolite channels, whereas the 1874 cm^-1 band is proposed to be induced by 2 nm FeAlO_x nano-particles. The ex-[Fe,Ga]MFI catalyst showed a similar absorption pattern (doublet), which is shifted to lower wavenumbers (1881 and 1867 cm^-1), suggesting that both frequencies are affected by the vicinity of Ga (or Al) to the Fe site involved. The absence of bands at 1765 and 1835 cm^-1 suggests that the isolated sites causing these absorptions are in the Fe^III state in ex-[Fe,Al]MFI and ex-[Fe,Ga]MFI. For the ex-[Fe]MFI sample, which did not contain any 2 nm FeO_x nano-particles, an NO absorption band at 1854 cm^-1 is assigned to mono-nitrosyl on extra-framework oligonuclear (Fe^IIO)_n species in the zeolite channels.     

New hypotheses for hydrogenase implication in the corrosion of mild steel

The influence of [Fe]-hydrogenase from Clostridium acetobutylicum was studied on the anaerobic corrosion of mild steel. Two short-circuited mild steel electrodes were exposed to the same solution and hydrogenase was retained on the surface of only one electrode thanks to a dialysis membrane. The galvanic current and the electrode potential were measured as a function of time in order to monitor the difference in electrochemical behaviour induced by the presence of hydrogenase. A sharp potential decrease of around 500 mV was controlled by the deoxygenating phase. When hydrogenase was introduced after complete deoxygenation, significant heterogeneous corrosion was observed under the vivianite deposit on the electrode in contact with hydrogenase, while the other electrode only showed the vivianite deposit, which was analysed by MEB and EDX. The effect of hydrogenase was then confirmed by monitoring the free potential of single coupons exposed or not to the enzyme in a classical cell after complete deoxygenating. In both phosphate and Tris-HCl buffers, the presence of hydrogenase increased the free potential around 60 mV and induced marked general corrosion. It was concluded that [Fe]-hydrogenase acts in the absence of any final electron acceptor by catalysing direct proton reduction on the mild steel surface.     

Characterization of phospholipase D activity in bovine photoreceptor membranes

Phospholipase D (E.C. 3.1.4.4.) was detected in isolated bovine rod outer segments (ROS) and its properties determined. The enzyme activity was assayed using either a sonicated microdispersion of 1,2-diacyl-sn-[2³H]glycerol-3-phosphocholine (PC), or [¹⁴C]ethanol. Using [³H]PC and ethanol as a substrate, we were able to detect the hydrolytic properties as well as the transphosphatidylation reaction catalyzed by phospholipase D (PLD): formation of [³H]phosphatidic acid and phosphatidylethanol [³H]PtdEt; whereas with [¹⁴C]ethanol or [³H]glycerol in the absence of exogenous PC, only transphosphatidylation reactions were detected (formation of [¹⁴C]PtdEt or [³H]phosphatidylglycerol, respectively). The use of varying concentrations of [³H]PC and 400 mM of ethanol gave an apparent K _m value for PC of 0.51 mM and a V _max value of 111 nmol × h⁻¹ × (mg protein)⁻¹. The activity was linear up to 60 min of incubation and up to 0.2 mg of protein. The optimal ethanol concentration was determined to be 400 mM, with an apparent K _m of 202 mM and a V _max value for ethanol of 125 nmol × h⁻¹ × (mg protein)⁻¹. A clear pH optimum was observed around 7. PLD activity was increased in the presence of 3-[(3-cholamidopropyl)dimethylammonio]-1-propane-sulfonate or sodium deoxycholate and inhibited with Triton X-100. The enzyme activity was also activated in the presence of Ca²⁺ or Mg²⁺ (1 mM) although these ions were not required for measuring PLD activity. The high specific activity of PLD found in purified ROS compared to the activity found in other subcellular fractions of the bovine retina suggests that this enzymatic activity is native to ROS. The present report is the first evidence of PLD activity associated with photoreceptor ROS.     

Catalytic oxidation of olefins with hydrogen peroxide catalyzed by [Fe(III)(salen)Cl] complex covalently linked to polyoxometalate

The catalytic activity of a hybrid compound [Fe(salen)-POM] (1) consisting of Fe(III)(salen)Cl [H₂salen = N,N′-bis(salicylidene)ethylenediamine] complex covalently linked to a Keggin type polyoxometalate, K₈SiW₁₁O₃₉, (POM) was studied in the oxidation of various olefins in acetonitrile, using hydrogen peroxide as oxygen source. While, [Fe(salen)-POM] catalyst showed moderate to good catalytic activity and product selectivity in the oxidation reactions, the complex Fe(III)(salen)Cl showed poor catalytic activity in these reactions. The effect of other parameters such solvent, oxidant, temperature and the metal type in Schiff base complex were also investigated.     

Copper is a Cofactor of the Formylglycine‐Generating Enzyme

Formylglycine‐generating enzyme (FGE) is an O₂‐utilizing oxidase that converts specific cysteine residues of client proteins to formylglycine. We show that Cu^I is an integral cofactor of this enzyme and binds with high affinity (K_D=of 10^?17 m ) to a pair of active‐site cysteines. These findings establish FGE as a novel type of copper enzyme.     

Electromechanical Properties of Acceptor‐Doped Lead‐Free Piezoelectric Ceramics

We have studied the processing and electromechanical properties of Mn and Fe‐doped 0.88[Bi_0.5Na_0.5TiO₃]–0.08[Bi_0.5K_0.5TiO₃]–0.04[Bi_0.5Li_0.5TiO₃] piezoelectric ceramics prepared by the mixed oxide route. Different amounts of Mn (0.01, 0.014, 0.015, 0.016, 0.017, 0.02, 0.022) or Fe (0.0125, 0.015, 0.0175) were doped to this lead‐free piezoelectric composition. Ceramics were sintered at different temperatures (1075°C–1150°C) to achieve the highest density and mechanical quality factor. Mn or Fe doping resulted in a considerable enhancement of Q_m in both planar and thickness resonance modes. In 1.5 mol% Mn‐doped ceramics sintered at 1100°C, a planar Q_m of about 970 and tanδ of 0.88% were obtained. In Fe‐doped ceramics, a planar Q_m as high as 900 was achieved. Acceptor dopants also resulted in decreasing the coupling coefficients, the piezoelectric charge coefficient, and the dielectric constant.     

Inhibition of cholesterol synthesis by cyclopropylamine derivatives of squalene in human hepatoblastoma cells in culture

Two squalene derivatives, trisnorsqualene cyclopropylamine and trisnorsqualeneN-methylcyclopropylamine, were synthesized and tested for inhibition of lanosterol and squalene epoxide formation from squalene in rat hepatic microsomes, and for the inhibition of cholesterol syntheses in human cultured hepatoblastoma (HepG2) cells. Trisnorsqualene cyclopropylamine inhibited [³H]-squalene conversion to [³H]squalene epoxide in microsomes (IC₅₀=5.0 μM), indicating that this derivative inhibited squalene mono-oxygenase. Trisnorsqualenen-methylcyclopropylamine inhibited [³H]squalene conversion to [³H]lanosterol (IC₅₀=12.0 μM) and caused [³H]-squalene epoxide to accumulate in microsomes, indicating that this derivative inhibited 2,3-oxidosqualene cyclase. Cholesterol biosynthesis from [¹⁴C]acetate in HepG2 cells was inhibited by both derivatives (IC₅₀=1.0 μM for trisnorsqualene cyclopropylamine; IC₅₀=0.5 μM for trisnorsqualeneN-methylcyclopropylamine). Cells incubated with trisnorsqualene cyclopropylamine accumulated [¹⁴C]squalene, while cells incubated with trisnorsqualeneN-methylcyclopropylamine accumulated [¹⁴C]squalene epoxide and [¹⁴C]squalene diepoxide. The concentration range of inhibitor which caused these intermediates to accumulate coincided with that which inhibited cholesterol synthesis. The results indicate that cyclopropylamine derivatives of squalene are effective inhibitors of cholesterol synthesis, and that substitutions at the nitrogen affect enzyme selectivity and thus the mechanism of action of the compounds.     

Effect of hyperpnea on enzymes of the CDPcholine path for phosphatidylcholine synthesis in rat lung

We have examined the activity of three enzymes in pulmonary surfactant phosphatidylcholine synthesis following the hyperpnea induced by having rats either inspire 5% CO₂/13%O₂/82% N₂ for 24 hr or swim in thermoneutral water for 30 min. Both stimuli markedly increase frequency and tidal volume of breathing and promote the release of surfactant. Lungs were perfused to remove blood, lavaged, and then homogenized in 1 mM Hepes, 0.15M KCl at pH 7.0. The homogenate was centrifuged at 9,000 g (av) for 10 min to sediment the mitochondria and lamellar bodies and at 100,000 g (av) for 60 min to obtain the microsomal and cytosol fractions. Incubations were carried out under determined optimal conditions and zero order kinetics. Choline kinase (CK), cholinephosphate cytidylytransferase (CP-cyT) and choline phosphotransferase (CPT) were assayed by the incorporation of [methyl-¹⁴C] choline chloride into phosphocholine, [methyl-¹⁴C]phosphocholine into CDPcholine, and [¹⁴C]CDPcholine into phosphatidylcholine, respectively. The incubation products were separated by thin-layer chromatography. Whereas both forms of hyperpnea increased the activity of CP-cyT in the microsomal fraction, they had no effect on the activity of either cytosolic CP-cyT and CK, or microsomal CPT. A similar increase in tidal volume in an isolated perfused rat lung had no effect. We conclude that,in vivo, hyperpnea increases the activity of CP-cyT, the rate-limiting enzyme in phosphatidylcholine synthesis. Whether this is due to an increase in the amount of enzyme, or of a cofactor, is unknown.     

The frataxin homologue Fra plays a key role in intracellular iron channeling in Bacillus subtilis

Frataxin homologues are important iron chaperones in eukarya and prokarya. Using a native proteomics approach we were able to identify the structural frataxin homologue Fra (formerly YdhG) of Bacillus subtilis and to quantify its native iron‐binding stoichiometry. Using recombinant proteins we could show in vitro that Fra is able to transfer iron onto the B. subtilis SUF system for iron–sulfur cluster biosynthesis. In a four‐constituents reconstitution system (including SufU, SufS, Fra and CitB) we observed a Fra‐dependent formation of a [4 Fe–4 S] cluster on SufU that could be efficiently transferred onto the target apo‐aconitase (CitB). A Δfra deletion mutant showed a severe growth phenotype associated with a broadly disturbed iron homeostasis; this indicates that Fra is a central component of intracellular iron channeling in B. subtilis.     

Heterologous Expression of an Unusual Ketosynthase,SxtA, Leads to Production of Saxitoxin Intermediates in Escherichia coli

Paralytic shellfish toxins (PSTs) are neurotoxic alkaloids produced by freshwater cyanobacteria and marine dinoflagellates. Due to their antagonism of voltage-gated sodium channels in excitable cells, certain analogues are of significant pharmacological interest. The biosynthesis of the parent compound, saxitoxin, is initiated with the formation of 4-amino-3-oxo-guanidinoheptane (ethyl ketone) by an unusual polyketide synthase-like enzyme, SxtA. We have heterologously expressed SxtA from Raphidiopsis raciborskii T3 in Escherichia coli and analysed its activity in vivo. Ethyl ketone and a truncated analogue, methyl ketone, were detected by HPLC-ESI-HRMS analysis, thus suggesting that SxtA has relaxed substrate specificity in vivo. The chemical structures of these products were further verified by tandem mass spectrometry and labelled-precursor feeding with [guanidino-¹⁵N₂] arginine and [1,2-¹³C₂] acetate. These results indicate that the reactions catalysed by SxtA could give rise to multiple PST variants, including analogues of ecological and pharmacological significance.     

Catalysis of an Essential Step in Vitamin B2 Biosynthesis by a Consortium of Broad Spectrum Hydrolases

An enzyme catalysing the essential dephosphorylation of the riboflavin precursor, 5‐amino‐6‐ribitylamino‐2,4(1H,3H)‐pyrimidinedione 5′‐phosphate ( 6 ), was purified about 800‐fold from a riboflavin‐producing Bacillus subtilis strain, and was assigned as the translation product of the ycsE gene by mass spectrometry. YcsE is a member of the large haloacid dehalogenase (HAD) superfamily. The recombinant protein was expressed in Escherichia coli. It catalyses the hydrolysis of 6 (v_max, 12 μmol mg^?1 min^?1; K_M, 54 μm ) and of FMN (v_max, 25 μmol mg^?1 min^?1; K_M, 135 μm ). A ycsE deletion mutant of B. subtilis was not riboflavin dependent. Two additional proteins (YwtE, YitU) that catalyse the hydrolysis of 6 at appreciable rates were identified by screening 13 putative HAD superfamily members from B. subtilis. The evolutionary processes that have resulted in the handling of an essential step in the biosynthesis of an essential cofactor by a consortium of promiscuous enzymes require further analysis.     

Exploring Electron/Proton Transfer and Conformational Changes in the Nitrogenase MoFe Protein and FeMo-cofactor Through Cryoreduction/EPR Measurements

We combine cryoreduction/annealing/EPR measurements of nitrogenase MoFe protein with results of earlier investigations to provide a detailed view of the electron/proton transfer events and conformational changes that occur during early stages of [e⁻/H⁺] accumulation by the MoFe protein. This includes reduction of: 1) the non-catalytic state of the iron-molybdenum cofactor (FeMo-co) active site that is generated by chemical oxidation of the resting-state cofactor (S=3/2) within resting MoFe (E₀); and 2) the catalytic state that has accumulated n=1 [e⁻/H⁺] above the resting-state level, denoted E₁(1H) (S≥1) in the Lowe-Thorneley kinetic scheme. FeMo-co does not undergo a major change of conformation during reduction of oxidized FeMo-co. In contrast, FeMo-co undergoes substantial conformational changes during the reduction of E₀ to E₁(1H), and of E₁(1H) to E₂(2H) (S=3/2). The experimental results further suggest that the E₁(1H)→E₂(2H) step involves coupled delivery of a proton and an electron (PCET) to FeMo-co of E₁(H) to generate a nonequilibrium S= form E₂(2H)*. This subsequently undergoes conformational relaxation and attendant change in the FeMo-co spin state, to generate the equilibrium E₂(2H) (S=3/2) state. Unexpectedly, these experiments also reveal conformational coupling between FeMo-co and the P cluster, and between the Fe protein binding and FeMo-co, which might play a role in gated electron transfer from reduced Fe protein to FeMo-co.     

METAL EXTRACTION BY ALKYL SUBSTITUTED DIPHOSPHONIC ACIDS. PART 4 P,P′-DI(2-ETHYLHEXYL) BUTANEDIPHOSPHONIC ACID

ABSTRACT

As part of an ongoing investigation of the properties of dialkyl substituted diphosphonic acids as solvent extraction reagents for metal cations, we have studied the extraction of alkaline earth cations, Fe(III) and representative actinides (Am(III), U(VI) and Th(IV)) at tracer-level concentration by o-xylene solutions of P,P′-di(2-ethylhexyl) butanediphosphonic acid, H₂DEH[BuDP]. The extractant and acid dependencies of these metal ions exhibited significant differences from those of the previously investigated analogous extractants in which the two phosphonate groups are separated by a methylene or an ethylene bridge. The aggregation of H₂DEH[BuDP] was investigated in toluene at 25° C by vapor pressure osmometry. H₂DEH[BuDP] was found to exist predominantly as a trimeric species in the 0.1-0.005 molal concentration range. Osmometric measurements and infrared spectra indicate that Ca(II) is extracted into H₂DEH[BuDP] solutions with little disruption of the structure of the extractant. Iron(III) causes significant deprotonation of the ligand and dramatically changes the apparent aggregation number. A comparison of the extraction of Ca(II), Am(III) and Fe(III) by H₂DEH[BuDP] with data obtained using bis(2-ethylhexyl) phosphoric acid (HDEHP) or 2-ethylhexyl 2-ethylhexylphosphonic acid (HEH[EHP]) as the extractant indicates that H₂DEH[BuDP] has characteristics similar to these monofunctional analogs. Infrared spectra of the Ca(II) and Fe(III) salts of H₂DEH[BuDP] show a shift of both v_asym (POO?) and v_sym (POO?) to lower frequencies relative to their values in the sodium salt. This indicates a symmetrical interaction between the metal ion and the phosphonate groups through chelate and/or bridging interactions.