Heterometallic [AgFe3S4] ferredoxin variants: synthesis, characterization, and the first crystal structure of an engineered heterometallic iron–sulfur protein

Heterometallic [AgFe₃S₄] iron–sulfur clusters assembled in wild-type Pyrococcus furiosus ferredoxin and two variants, D14C and D14H, are characterized. The crystal structure of the [AgFe₃S₄] D14C variant shows that the silver(I) ion is indeed part of the cluster and is coordinated to the thiolate group of residue 14. Cyclic voltammetry shows one redox pair with a reduction potential of +220 mV versus the standard hydrogen electrode which is assigned to the [AgFe₃S₄]^2+/+ couple. The oxidized form of the [AgFe₃S₄] D14C variant is stable in the presence of dioxygen, whereas the oxidized forms of the [AgFe₃S₄] wild type and D14H variants convert to the [Fe₃S₄] ferredoxin form. The monovalent d ¹⁰ silver(I) ion stabilizes the [Fe₃S₄]^+/0 cluster fragment, as opposed to divalent d ¹⁰ metal ions, resulting in more than 0.4 V difference in reduction potentials between the silver(I) and, e.g., zinc(II) heterometallic [MFe₃S₄] ferredoxins. The trend in reduction potentials for the variants containing the [AgFe₃S₄] cluster is wild type ≤ D14C < D14H and shows the same trend as reported for the variants containing the [Fe₃S₄] cluster, but is different from the D14C < D14H < wild type trend reported for the [Fe₄S₄] ferredoxin. The similarity in the reduction potential trend for the variants containing the heterometallic [AgFe₃S₄] cluster and the [Fe₃S₄] cluster can be rationalized in terms of the electrostatic influence of the residue 14 side chains, rather than the dissociation constant of this residue, as is the case for [Fe₄S₄] ferredoxins. The trends in reduction potentials are in line with there being no electronic coupling between the silver(I) ion and the Fe₃S₄ fragment.     

Facile formation of a heterobimetallic cluster with a cubane-like [Mo3S4Cu] core

Reaction of the cluster salt [(η⁵-Cp^′)₃Mo₃S₄][pts] ([1][pts], Cp^′=methylcyclopentadienyl; pts=p-toluenesulfonate) with CuCl yielded a new heterobimetallic cluster, [(η⁵-Cp^′)₃Mo₃S₄Cu(Cl)][pts] ([2][pts]). X-ray crystal structure determination of [1][pts] and [2][pts] showed that the incorporation of CuCl into the Mo₃S₄ cluster core has only minor consequences on the Mo-Mo and Mo-S distances. The metal atoms in the cluster core of [2]⁺ form an almost regular tetrahedron. The [2]⁺ cation conforms to an idealized C_3v symmetry ignoring the Cp^′ groups since the Cu-Cl bond is almost aligned with the axis defined by the Cu-S(4) cube diagonal.     

Coordination diversity of N-phosphoryl-N′-phenylthiourea (LH) towards Co, Ni and Pd cations: Crystal structure of ML2-N,S and ML2-O,S chelates

Thiourea, PhNHC(S)NHP(O)(OPrⁱ)₂ (LH) chelates of Co^II, Ni^II, and Pd^II ions have been obtained and investigated by single-crystal X-ray diffraction, UV, IR, NMR spectroscopy, and EI mass-spectrometry. The unusual 1,3-N,S-coordination via sulfur and NP(O) nitrogen atoms has been found in the trans-square-planar NiL₂ and PdL₂ complexes, whereas the 1,5-O,S-coordination is realized in the tetrahedral CoL₂ complex. DFT calculations have revealed significant stabilization of the 1,3-N,S-structures due to stronger crystal field and the NH-OP hydrogen bonds.     

Infrared spectroscopic studies of aqueous solutions of dioxouranium(VI) and its hydrolysed products and of in situ electro-generated dioxouranium(V)

Aqueous solutions of dioxouranium(VI) (pH range 0 to 4) give rise to bands at 954 and 938 cm⁻¹ attributable to the v₃(MO₂) stretching modes of the UO₂²⁺ and (UO₂)₂(OH)_2²⁺ cations, respectively. A shoulder at 916 cm⁻¹ is assigned to the v₃(MO₂) mode of hydrolysed dioxouranium(VI) species of higher nuclearity. Infrared spectro-electrochemical studies using a thin-layer reflection-absorption cell have facilitated the study of the reduction of aqueous solutions of dioxouranium(VI) to yield dioxouranium(V) which may be further reduced to uranium(IV). The electrogeneration of dioxouranium(V) is monitored by following the increase in intensity of a band at 914 cm⁻¹ which is present in the spectra at potentials between −0.2 and −0.8 V. The dioxouranium(V) species is predominantly in the form UO₂⁺, which may be in solution or incorporated into an insoluble phase of uranium oxides which deposit onto the working electrode. The U^VO bond length is estimated to be 1.76 Å, 0.03 Å longer than the U^VIO bond in aqueous solution. The maximum concentration of UO₂⁺able to be achieved is highly dependent on the pH and is optimum at pH 3.4. Changes in the pH of the solution under study can be monitored by infrared spectroscopy during the course of the reduction by determining the relative concentrations of hydrolysed dioxouranium(VI) species.     

Synthesis and characterization of uranyl(VI) and thorium(IV) complexes with phosphine oxides. The crystal structure of UO2(NO3)2(NO2-C6H4Ph2PO)2

Uranyl(VI) and thorium(IV) complexes of the type UO₂(NO₃)₂(L¹)₂, UO₂(NO₃)₂(L²)₂, UO₂(CH₃COO)₂L¹, UO₂(CH₃COO)₂L², Th(NO₃)₄(L¹)₂ and Th(NO₃)₄(L²)₂ (L¹ = (2-nitro)phenyl-bis-phenyl phosphine oxide, L² = triferrocenylphosphine oxide) are reported, together with their physico-chemical properties.The crystal structure of UO₂(NO₃)₂(L¹)₂ is also reported. The crystals are monoclinic, space group P2₁/n with a = 17.78(1), b = 13.88(1), c = 17.37(1) Å, β = 114.8(1)° for Z = 4. The uranium atom is 8-coordinated, the uranyl(VI) group being equatorially surrounded by an irregular hexagon of six oxygen atoms from two trans neutral ligands and two nitrato groups.     

Investigation of the thermal unfolding of secondary and tertiary structure in E. coli tRNAfMet by high-resolution nmr

The high-resolution (300 MHz) proton nmr spectrum of E. coli tRNA^fMet has been examined in 0.17M NaCl, with and without Mg²⁺, and at various temperatures. In light of recent studies of other E. coli tRNA and fragments of tRNA^fMet, some low field (11–15 ppm) resonances previously assigned to secondary structure base pairs are reassigned to a tertiary structure A₁₄–S⁴U₈ base pair and a protected uridine residue in the anticodon loop. These two resonances and other low field resonances which are assigned to secondary structure base pairs are used to monitor the thermal unfolding of the molecule. In the absence of Mg²⁺ the tertiary structure base pair is present only to ～45°C, but in the presence of Mg²⁺ it remains until at least 70°C. Analysis of the temperature dependence of other low field resonances indicates that the melting of the dihydrouridine stem occurs more or less simultaneously with the loss of tertiary structure. The observation of the resonance from the A₁₄–S⁴U₈ base pair proves that tertiary structure is present in this molecule below 40°C, even in the absence of Mg²⁺.     

Mechanism of protonation of the over-reduced Mn4CaO5 cluster in photosystem II

Based on characterization by X-ray absorption spectroscopy, it has been proposed that the Mn₄CaO₅ cluster in the crystal structure of the water-oxidizing enzyme, photosystem II (PSII), may represent an over-reduced form arising from reduction by the X-ray beam. Using a quantum mechanical/molecular mechanical approach, and assuming that all of the μ-oxo bridges are deprotonated in S₁, we analyzed the reduction process of the Mn₄CaO₅ cluster. In the crystal structure, the O atom (O5), which is linked with three Mn atoms and one Ca atom, has no H-bond. When reduced to S_–2, unexpectedly, a water molecule at Ca²⁺ (W3) reoriented itself, formed a H-bond with O5, and released a proton to O5, resulting in formation of OH⁻ at both W3 and O5. Once generated, the OH⁻ group at O5 was stable, because the W3…O5 H-bond had already disappeared. A weak binding of H₂O at Ca²⁺ led W3 to reorient and serve as a proton donor to O5 upon over-reduction.     

Coordination chemistry of lanthanides with cryptands. An X-ray and spectroscopic study of the complex Nd2(NO3)6 [C18H36O6N2]·H2O

By reacting neodymium nitrate hexahydrate with the cryptand 〈222〉 in methanol, the complex Nd₂-(NO₃)₆[C₁₈H₃₆O₆N₂]·H₂O was obtained and analyzed by single-crystal X-ray diffraction. The cell is triclinic P$\text{1}$ with a = 14.870(2) Å, b = 13.261(2) Å, c = 8.832(1) Å, α = 91.2(1)°, β = 93.4(1)°, γ = 87.6(1)°, Z = 2 and U = 1736.6 ų. The structure was refined by least-squares methods to the conventional R = 0.039 for 6177 observed reflections. The compound contains the cations [Nd〈222〉(NO₃)]²⁺ and the anions [Nd(NO₃)₅·H₂O]^2?, and is isostructural with the samarium analogue. Solid state fluorescence spectra of the title complex were measured at room and liquid nitrogen temperature, and the transitions ⁴F_3/2 → ⁴I_9/2 and ⁴F_3/2 → ⁴I_11/2 analyzed.     

Synthesis, structure and reactivity of complexes containing [M(S2)2] fragments (M=Ru, Os; (S2)=1,2-benzenedithiolate (2−))

Subsequent addition of 1,2-benzenedithiol (S₂-H₂) and nBuLi to a solution of [Ru(NO)Cl₃ · xMeOH] in THF afforded exclusively the monomeric species NBu₄[Ru^II(NO)(S₂)₂] (1). Formation of dimeric (NBu₄)₂[Ru^II(NO)(S₂)₂]₂ (2) has been confirmed when the deprotonated ligand S₂-Li₂ was added to [Ru(NO)Cl₃ · xMeOH] and allowed to stir for 30 h. The monomer 1 undergoes aerial oxidation to give (NBu₄)₂[Ru^IV(S₂)₃] (3). The reaction between RuCl₃ · xH₂O and S₂-H₂ in the presence of NaOMe, afforded the dinulear Ru^III species (NMe₄)₂[Ru^III(S₂)₂]₂ (4). A modified method for the preparation of 1 is being employed to synthesize the osmium analogue NBu₄[Os(NO)(S₂)₂] (5) effectively. The solid state structures of 1, 2 and 3 were determined by X-ray crystal structure analysis. A comparison of relevant bond distance data suggests that 1,2-benzenedithiolate acts as an “innocent” ligand.     

A relativistic DFT probe of polypyrrolic macrocyclic diuranium(III) complexes with terminal solvents and iodines

Relativistic density functional theory finds that two isomers of a diuranium(III) complex of a polypyrrolic macrocycle (H₄L) feature active sites on uranium moieties, allowing for their potential application in activating industrially and economically important small molecules. To address this, a series of adducts [(X)_nU₂(L)]^(2–m)+ (X?=?THF, I^? and HI; n?=?1 and 2; m?=?0, 1 and 2) have been examined. The coordination from X to the exposed uranium(s) changes the general geometry and electronic structure slightly. Thermodynamic calculations reveal that iodine termination is energetically favored over THF/HI coordination.

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Graphical abstract Scalar and spin-orbit coupling relativistic DFT calculation reveals that the active sites on the uranium moieties of [U₂(L)]²⁺ lead to formation of adducts [(THF)_nU₂(L)]²⁺, [I_nU₂(L)]^(2–n)+ and [(HI)_nU₂(L)]²⁺ (n?=?1 and 2). Coordination to the exposed uranium(s) changes geometrical and electronic properties slightly, but iodine termination is the most energetically favored.

     

Characterization of ribosomes from the seed of Pinus lambertiana

Ribosomes isolated from seeds of the sugar pine, Pinus lambertiana, have been characterized: The ribosome has a sedimentation coefficient (s_20,w⁰) of 78·2 S and contains 41 % RNA and 58 % protein. On dialysis against buffer containing 0·5-1 mM MgCl₂, the ribosome was reversibly transformed into an intermediate form (60 S). Further removal of Mg²⁺ causes the intermediate ribosome to dissociate into subunits (30 S and 40 S). Treatment of the intermediate ribosome with p-chloromercuribenzoic acid caused the dissociation of the particle into subunits. Incubating the 80 S ribosome with the sulfhydryl reagent caused a rapid transformation of the particle into an intermediate type particle. These results suggest that sulfhydryl groups are involved not only in associating the subunits but also in maintaining the compact structure of the ribosomes. The ribosome contains three ribosomal RNA components of 28 S, 18 S and 5 S. The base compositions of the three ribosomal RNA components are different.     

Synthesis and crystal structure of the low-spin iron(II) complex [Fe(bpz)3](ClO4)2 · H2O (bpz=2,2-bipyrazine)

The crystal structure of the title compound [Fe(bpz)₃](ClO₄)₂ · H₂O (bpz=2,2^′-bipyrazine) has been determined by a single crystal X-ray diffraction study at 293(2) K. The complex is monoclinic, P2₁/c, a=17.263(3), b=9.983(2), c=17.921(4) Å, β=107.94(3)°, V=2938.3(10) ų, Z=4, R=0.073 and R_w=0.118. The structure is made up of tris-chelated [Fe(bpz)₃]²⁺ cations, uncoordinated perchlorate anions and crystallization water molecules. The iron atom exhibits a FeN₆ distorted octahedral geometry with average Fe-N bond length and N-Fe-N bidentate angle of 1.962(5) Å and 81.6(2)°. The value of the Fe-N bond distance and that of the room temperature magnetic moment are in agreement with a singlet ¹A₁ ground state. The structure of 1 is compared to those of other tris-chelated iron(II) complexes with bidentate nitrogen heterocycles.     

Novel Processes for Anaerobic Sulfate Production from Elemental Sulfur by Sulfate-Reducing Bacteria

Sulfate reducers and related organisms which had previously been found to reduce Fe(III) with H₂ or organic electron donors oxidized S⁰ to sulfate when Mn(IV) was provided as an electron acceptor. Organisms catalyzing this reaction in washed cell suspensions included Desulfovibrio desulfuricans, Desulfomicrobium baculatum, Desulfobacterium autotrophicum, Desulfuromonas acetoxidans, and Geobacter metallireducens. These organisms produced little or no sulfate from S⁰ with Fe(III) as a potential electron acceptor or in the absence of an electron acceptor. In detailed studies with Desulfovibrio desulfuricans, the stoichiometry of sulfate and Mn(II) production was consistent with the reaction S⁰ + 3 MnO₂ + 4H⁺→SO₄^2- + 3Mn(II) + 2H₂O. None of the organisms evaluated could be grown with S⁰ as the sole electron donor and Mn(IV) as the electron acceptor. In contrast to the other sulfate reducers evaluated, Desulfobulbus propionicus produced sulfate from S⁰ in the absence of an electron acceptor and Fe(III) oxide stimulated sulfate production. Sulfide also accumulated in the absence of Mn(IV) or Fe(III). The stoichiometry of sulfate and sulfide production indicated that Desulfobulbus propionicus disproportionates S⁰ as follows: 4S⁰ + 4H₂O→SO₄^2- + 3HS^- + 5 H⁺. Growth of Desulfobulbus propionicus with S⁰ as the electron donor and Fe(III) as a sulfide sink and/or electron acceptor was very slow. The S⁰ oxidation coupled to Mn(IV) reduction described here provides a potential explanation for the Mn(IV)-dependent sulfate production that previous studies have observed in anoxic marine sediments. Desulfobulbus propionicus is the first example of a pure culture known to disproportionate S⁰.     

Single molecule magnet: Heterodinuclear cyano-bridged cubic cluster [(Tp)8Fe4Ni4(CN)12] (Tp = hydrotris(1-pyrazolyl)borate)

The octanuclear cyano-bridged cluster [(Tp)₈Fe₄Ni₄(CN)₁₂] · H₂O · 24CH₃CN (1) (Tp = hydrotris(1-pyrazolyl)borate) showing magnetic properties of single-molecule magnet has been synthesized by reaction of [fac-Fe(Tp)(CN)₃]⁻ with {(Tp)Ni(NO₃)} species formed from an equimolar reaction mixture of Ni(NO₃)₂ · 6H₂O and KTp in MeCN. The X-ray analysis of 1 shows molecular cube structure in which Fe^III and Ni^II ions reside in alternate corners. The average intramolecular Fe?Ni distance is 5.124 Å. Out-of-phase ac susceptibility and reduce magnetization measurements show that 1 is a single molecule magnet with ground spin state S = 6 and spin reversal energy barrier U = 14 K. Magnetic hysteresis loops were also observed by applying fast sweeping field.     

Photolysis of the ion pair [Pt(NH3)5Cl]S2O8: reduction of platinum(IV) induced by outer-sphere charge transfer excitation

The ion pair [Pt^IV(NH₃)₅Cl]³⁺S₂O₈²⁻ shows a S₂O₈²⁻ → [Pt(NH₃)₅Cl]³⁺ outer-sphere charge transfer (OSCT) absorption at λ_max=267 nm. OSCT excitation leads to the reduction of Pt(IV) by S₂O₈²⁻ to Pt(II) with φ=3×10⁻³ at λ_irr=280 nm.     

Structures, spectroscopic and thermodynamic properties of U2On (n = 0 ∼ 2, 4) molecules: a density functional theory study

The equilibrium structures, spectroscopic and thermodynamic parameters [entropy (S), internal energy (E), heat capacity (C _p)] of U₂, U₂O, U₂O₂ and U₂O₄ uranium oxide molecules were investigated systematically using density functional theory (DFT). Our computations indicated that the ground electronic state of U₂ is the septet state and the equilibrium bond length is 2.194 Å; the ground electronic state of U₂O and U₂O₂ were found to be $ {\tilde{X}}^3\varPhi $ and $ {\tilde{X}}^3{\sum}_{\mathrm{g}} $ with stable C _∞v and D _∞h linear structures, respectively. The bridge-bonded structure with D _2h symmetry and $ {\tilde{X}}^3{\mathrm{B}}_{1\mathrm{g}} $ state is the most stable configuration for the U₂O₄ molecule. Mulliken population analyses show that U atoms always lose electrons to become the donor and O atoms always obtain electrons as the acceptor. Molecular orbital analyses demonstrated that the frontier orbitals of the title molecules were contributed mostly by 5f atomic orbitals of U atoms. Vibrational frequencies analyses indicate that the maximum absorption peaks stem from the stretching mode of U–O bonds in U₂O, U₂O₂ and U₂O₄. In addition, thermodynamic data of U₂O_n (n?=?0?～?4) molecules at elevated temperatures of 293.0 K to 393.0 K was predicted.     

Cu(II) Schiff-base complex with [N3O] binding site and a pendant S-methylisothiosemicarbazide arm

A neutral four-coordinate Cu^IIN₃O complex with Shiff base ligand, product of template assembling of two S-methylisothiosemicarbazide units with benzoylacetone and nitromalondialdehyde, was synthesized by two steps procedure via Cu^IIN₂O₂ chelate precursor and was fully characterized by elemental analysis, IR, UV-Vis, ESR spectra and magnetochemistry. X-ray analysis confirms its structural assignment. The assembling ligand assumes an acyclic structure of ‘4+1’ type where a non-bonding interaction between the metal and the amino group of one S-methylisothiosemicarbazide unit takes place.     

The low spin - high spin equilibrium in the S2-state of the water oxidizing enzyme

In Photosystem II (PSII), the Mn₄CaO₅-cluster of the active site advances through five sequential oxidation states (S₀ to S₄) before water is oxidized and O₂ is generated. Here, we have studied the transition between the low spin (LS) and high spin (HS) configurations of S₂ using EPR spectroscopy, quantum chemical calculations using Density Functional Theory (DFT), and time-resolved UV-visible absorption spectroscopy. The EPR experiments show that the equilibrium between S₂^LS and S₂^HS is pH dependent, with a pK_a?≈?8.3 (n?≈?4) for the native Mn₄CaO₅ and pK_a?≈?7.5 (n?≈?1) for Mn₄SrO₅. The DFT results suggest that exchanging Ca with Sr modifies the electronic structure of several titratable groups within the active site, including groups that are not direct ligands to Ca/Sr, e.g., W1/W2, Asp61, His332 and His337. This is consistent with the complex modification of the pK_a upon the Ca/Sr exchange. EPR also showed that NH₃ addition reversed the effect of high pH, NH₃-S₂^LS being present at all pH values studied. Absorption spectroscopy indicates that NH₃ is no longer bound in the S₃Tyr_Z state, consistent with EPR data showing minor or no NH₃-induced modification of S₃ and S₀. In both Ca-PSII and Sr-PSII, S₂^HS was capable of advancing to S₃ at low temperature (198?K). This is an experimental demonstration that the S₂^LS is formed first and advances to S₃via the S₂^HS state without detectable intermediates. We discuss the nature of the changes occurring in the S₂^LS to S₂^HS transition which allow the S₂^HS to S₃ transition to occur below 200?K. This work also provides a protocol for generating S₃ in concentrated samples without the need for saturating flashes.     

Lanthanide ions induce hydrolysis of hemoglobin-bound 2,3-diphosphoglycerate (2,3-DPG), conformational changes of globin and bidirectional changes of 2,3-DPG-hemoglobin’s oxygen affinity

The changes in structure and function of 2,3-diphosphoglycerate-hemoglobin (2,3-DPG-Hb) induced by Ln³⁺ binding were studied by spectroscopic methods. The binding of lanthanide cations to 2,3-DPG is prior to that to Hb. Ln³⁺ binding causes the hydrolysis of either one from the two phosphomonoester bonds in 2,3-DPG non-specifically. The results using the ultrafiltration method indicate that Ln³⁺ binding sites for Hb can be classified into three categories: i.e. positive cooperative sites (N_I), non-cooperative strong sites (N_S) and non-cooperative weak sites (N_W) with binding constants in decreasing order: K_I>K_S>K_W. The total number of binding sites amounts to about 65 per Hb tetramer. Information on reaction kinetics was obtained from the change of intrinsic fluorescence in Hb monitored by stopped-flow fluorometry. Fluctuation of fluorescence dependent on Ln³⁺ concentration and temperature was observed and can be attributed to the successive conformational changes induced by Ln³⁺ binding. The results also reveal the bidirectional changes of the oxygen affinity of Hb in the dependence on Ln³⁺ concentration. At the range of [Ln³⁺]/[Hb]<2, the marked increase of oxygen affinity (P₅₀ decrease) with the Ln³⁺ concentration can be attributed to the hydrolysis of 2,3-DPG, while the slight rebound of oxygen affinity in higher Ln³⁺ concentration can be interpreted by the transition to the T-state of the Hb tetramer induced by Ln³⁺ binding. This was indicated by the changes in secondary structure characterized by the decrease of α-helix content.     

Use of a cubane-type Mo3CoS4 molecular cluster as paramagnetic unit in the synthesis of hybrid charge-transfer salts

Selective substitution of the chlorine atom coordinated to cobalt in the paramagnetic Mo₃(CoCl)S₄(dmpe)₃Cl₃ (dmpe = 1,2-bis(dimethylphosphanyl) ethane) complex with a S = 1/2 ground state has been achieved by iodine oxidation to afford the also paramagnetic [Mo₃(CoI)S₄(dmpe)₃Cl₃]I ([1]I) salt with a S = 1 ground state in almost quantitative yield. Replacement of chorine by iodine has no significant effect on the structural and electrochemical properties of the Mo₃CoS₄ system. Metathesis of the [1]I salt with the paramagnetic nickel anionic dithiolate [Ni(mnt)₂]⁻ (mnt = maleonitrilodithiolate) affords [1]₂[Ni(mnt)₂]. The stoichiometry evidenced by X-ray analysis reveals that reduction of the [Ni(mnt)₂]⁻ radical to the corresponding diamagnetic closed shell [Ni(mnt)₂]²⁻ dianion, presumably via dismutation, has occurred during the metathesis process. The crystal structure of [1]₂[Ni(mnt)₂] consists of [Ni(mnt)₂]²⁻ dianions sandwiched by two cluster 1⁺ cations which yield {1⁺·[Ni(mnt)₂]²⁻·1⁺} subunits arranged along the crystallographic c axis. Magnetic susceptibility measurements for [1]₂[Ni(mnt)₂] show a χT product of 0.99 emu K/mol largely unchanged in the 10-300 K range. This behavior agrees with the presence of an S = 1 cluster 1⁺ cation while the Ni(mnt)₂ moiety does not contribute to the paramagnetism of the sample.