Visualising the carboxylate shift at a bioinspired diiron(II) site in the solid state

A novel pyrazolate-based diiron(II) complex shows five different binding modes of exogenous carboxylate ligands in a single crystal structure. Temperature dependent X-ray data reveal thermally induced disorder due to carboxylate dynamics that resemble the carboxylate shift, as it is known from various diiron enzyme active sites.     

Heme/non-heme diiron(II) complexes and O2, CO, and NO adducts as reduced and substrate-bound models for the active site of bacterial nitric oxide reductase

As a first generation model for the reactive reduced active-site form of bacterial nitric oxide reductase, a heme/non-heme diiron(II) complex [(6L)Fe(II)...Fe(II)-(Cl)]+ (2) {where 6L = partially fluorinated tetraphenylporphyrin with a tethered tetradentate TMPA chelate; TMPA = tris(2-pyridyl)amine} was generated by reduction of the corresponding mu-oxo diferric compound [(6L)Fe(III)-O-Fe(III)-Cl]+ (1). Coordination chemistry models for reactions of reduced NOR with O2, CO, and NO were also developed. With O2 and CO, adducts are formed, [(6L)Fe(III)(O2-))(thf)...Fe(II)-Cl]B(C6F5)4 (2a x O2) {lambda(max) 418 (Soret), 536 nm; nu(O-O) = 1176 cm(-1), nu(Fe-O) = 574 cm(-1) and [(6L)Fe(II)(CO)(thf)Fe(II)-Cl]B(C6F5)4 (2a x CO) {nu(CO) 1969 cm(-1)}, respectively. Reaction of purified nitric oxide with 2 leads to the dinitrosyl complex [(6L)Fe(NO)Fe(NO)-Cl]B(C6F5)4 (2a x (NO)2) with nu(NO) absorptions at 1798 cm(-1) (non-heme Fe-NO) and 1689 cm(-1) (heme-NO).     

DFT calculations for intermediate and active states of the diiron center with a tryptophan or tyrosine radical in Escherichia coli ribonucleotide reductase

Class Ia ribonucleotide reductase subunit R2 contains a diiron active site. In this paper, active-site models for the intermediate X-Trp48(?+) and X-Tyr122(?), the active Fe(III)Fe(III)-Tyr122(?), and the met Fe(III)Fe(III) states of Escherichia coli R2 are studied, using broken-symmetry density functional theory incorporated with the conductor-like screening solvation model. Different structural isomers and different protonation states have been explored. Calculated geometric, energetic, Mo?ssbauer, hyperfine, and redox properties are compared with available experimental data. Feasible detailed structures of these intermediate and active states are proposed. Asp84 and Trp48 are most likely the main contributing residues to the result that the transient Fe(IV)Fe(IV) state is not observed in wild-type class Ia E. coli R2. Asp84 is proposed to serve as a proton-transfer conduit between the diiron cluster and Tyr122 in both the tyrosine radical activation pathway and the first steps of the catalytic proton-coupled electron-transfer pathway. Proton-coupled and simple redox potential calculations show that the kinetic control of proton transfer to Tyr122(?) plays a critical role in preventing reduction from the active Fe(III)Fe(III)-Tyr122(?) state to the met state, which is potentially the reason why Tyr122(?) in the active state can be stable over a very long period.     

Two CO molecules can bind concomitantly at the diiron site of NO reductase from Bacillus azotoformans

CO complexes formed in reduced nitric oxide reductase from Bacillus azotoformans were investigated with resonance Raman and FTIR techniques. These experiments shows the presence of two nu(C-O) bands, one at approximately 1970 cm-1 assigned to the heme-CO complex, and one at approximately 2070 cm-1 from the non-heme iron, FeBCO. At cryogenic temperatures, the heme-CO complex adopts a semi-bridging configuration with FeB which decreases its stretching frequency to approximately 1910 cm-1 and decreases the nu(C-O) of FeBCO by approximately 20 cm-1. The concomitant binding of two CO molecules, one per iron(II) at the active site, is consistent with the formation of a [{FeNO}7]2 iron-nitrosyl dimer during substrate turnover. This study strongly supports the notion that this family of enzymes utilizes a reaction mechanism based on catalysis by proximity, where the formation of two iron-nitrosyl groups promotes N-N bond formation.     

A density functional evaluation of an Fe(III)-Fe(IV) model diiron cluster: comparisons with ribonucleotide reductase intermediate X

Using broken-symmetry density functional theory and spin-projection methods, we have examined the electronic structure and properties of a large mixed-valent Fe(III)-Fe(IV) diiron system that displays two bidentate carboxylates and a single mu-oxo moiety as bridging ligands. Two carboxylates and a single oxygen species have long been implicated as core elements of the elusive intermediate X in ribonucleotide reductase. Spectroscopic studies of X have also identified the presence of an additional terminal or bridging oxygen-based ligand. Introduction of a second oxygen and protonated variants thereof in the core of our structural model is favored as a bridging hydroxide based on the lowest energy structure. M?ssbauer measurements indicate clearly that the two iron sites of X are distinct and that there is significant electron delocalization onto the oxygen-based ligands. For several examined spin states of our model cluster, M?ssbauer parameters from density functional calculations are neither able to differentiate between the iron sites nor reproduce the strong spin delocalization onto the oxygen-based ligands observed experimentally. The combined comparison of the calculated geometries, spin states, spin densities, and M?ssbauer properties for our model clusters with available experimental data for X implies that intermediate X is significantly different from the diiron structural models examined herein.     

A non-radical mechanism for methane hydroxylation at the diiron active site of soluble methane monooxygenase

We propose a non‐radical mechanism for the conversion of methane into methanol by soluble methane monooxygenase (sMMO), the active site of which involves a diiron active center. We assume the active site of the MMOH_Q intermediate, exhibiting direct reactivity with the methane substrate, to be a bis(μ‐oxo)diiron(IV ) complex in which one of the iron atoms is coordinatively unsaturated (five‐coordinate). Is it reasonable for such a diiron complex to be formed in the catalytic reaction of sMMO? The answer to this important question is positive from the viewpoint of energetics in density functional theory (DFT) calculations. Our model thus has a vacant coordination site for substrate methane. If MMOH_Q involves a coordinatively unsaturated iron atom at the active center, methane is effectively converted into methanol in the broken‐symmetry singlet state by a non‐radical mechanism; in the first step a methane C? H bond is dissociated via a four‐centered transition state (TS1) resulting in an important intermediate involving a hydroxo ligand and a methyl ligand, and in the second step the binding of the methyl ligand and the hydroxo ligand through a three‐centered transition state (TS2) results in the formation of a methanol complex. This mechanism is essentially identical to that of the methane–methanol conversion by the bare FeO⁺ complex and relevant transition metal–oxo complexes in the gas phase. Neither radical species nor ionic species are involved in this mechanism. We look in detail at kinetic isotope effects (KIEs) for H atom abstraction from methane on the basis of transition state theory with Wigner tunneling corrections.     

Synthesis and spectroscopy of micro-oxo (O(2)(-))-bridged heme/non-heme diiron complexes: models for the active site of nitric oxide reductase

In this paper, we describe the synthesis and study of a series of heme/non-heme Fe-O-Fe' complexes supported by a porphyrin and the tripodal nitrogen ligand TMPA [TMPA = tris(2-pyridylmethyl)amine]. The complete synthesis of [((6)L)Fe-O-Fe(X)](+) (1) (X = OMe(-) or Cl(-), 69:31 ratio), where (6)L is the dianion of 5-(o-O-[(N,N-bis(2-pyridylmethyl)-2-(6-methoxyl)pyridinemethanamine)phenyl]-10,15,20-tris(2,6-difluorophenyl)porphine, is reported. The crystal structure for 1.PF(6) reveals an intramolecular heme/non-heme diferric complex bridged by an Fe-O-Fe' moiety; 90 degree angle (Fe-O-Fe') = 166.7(3) degrees, and d(Fe.Fe') = 3.556 A. Crystal data for C(70)H(57)ClF(12)Fe(2)N(8)O(3)P (1.PF(6)): triclinic, Ponemacr;, a = 13.185(3) A, b = 14.590 (3) A, c = 16.885(4) A, alpha = 104.219(4) degrees, beta = 91.572(4) degrees, gamma = 107.907(4) degrees, V = 2977.3(11) A(3), Z = 2, T = 150(2) K. Complex 1 (where X = Cl(-)) is further characterized by UV-vis (lambda(max) = 328, 416 (Soret), 569 nm), (1)H NMR (delta 27-24 [TMPA -CH(2)-], 16.1 [pyrrole-H], 15.2-10.5 [PY-3H, PY-5H], 7.9-7.2 [m- and p-phenyl-H], 6.9-5.8 [PY-4H] ppm), resonance Raman (nu(as)(Fe-O-Fe') 844 cm(-)(1)), and M?ssbauer (delta(Fe) = 0.47, 0.41 mm/s; deltaE(A) = 1.59, 0.55 mm/s; 80 K) spectroscopies, MALDI-TOF mass spectrometry (m/z 1202), and SQUID susceptometry (J = - 114.82 cm(-)(1), S = 0). We have also synthesized a series of 3-, 4-, and 5-methyl-substituted as well as selectively deuterated TMPA(Fe') complexes and condensed these with the hydroxo complex (F(8))FeOH or (F(8)-d(8))FeOH to yield "untethered" Fe-O-Fe' analogues. Along with selective deuteration of the methylene hydrogens in TMPA, complete (1)H NMR spectroscopic assignments for 1 have been accomplished. The magnetic properties of several of the untethered complexes and a comparison to those of 1 are also presented. Complex 1 and related species represent good structural and spectroscopic models for the heme/non-heme diiron active site in the enzyme nitric oxide reductase.     

Mechanistic implications for the formation of the diiron cluster in ribonucleotide reductase provided by quantitative EPR spectroscopy

The small subunit of Escherichia coli ribonucleotide reductase (R2) is a homodimeric (betabeta) protein, in which each beta-peptide contains a diiron cluster composed of two inequivalent iron sites. R2 is capable of reductively activating O(2) to produce a stable tyrosine radical (Y122*), which is essential for production of deoxyribonucleotides on the larger R1 subunit. In this work, the paramagnetic Mn(II) ion is used as a spectroscopic probe to characterize the assembly of the R2 site with EPR spectroscopy. Upon titration of Mn(II) into samples of apoR2, we have been able to quantitatively follow three species (aquaMn(II), mononuclear Mn(II)R2, and dinuclear Mn(2)(II)R2) and fit each to a sequential two binding site model. As previously observed for Fe(II) binding within apoR2, one of the sites has a greater binding affinity relative to the other, K(1) = (5.5 +/- 1.1) x 10(5) M(-)(1) and K(2) = (3.9 +/- 0.6) x 10(4) M(-)(1), which are assigned to the B and A sites, respectively. In multiple titrations, only one dinuclear Mn(2)(II)R2 site was created per homodimer of R2, indicating that only one of the two beta-peptides of R2 is capable of binding Mn(II) following addition of Mn(II) to apoR2. Under anaerobic conditions, addition of only 2 equiv of Fe(II) to R2 (Fe(2)(II)R2) completely prevented the formation of any bound MnR2 species. Upon reaction of this sample with O(2) in the presence of Mn(II), both Y122* and Mn(2)(II)R2 were produced in equal amounts. Previous stopped-flow absorption spectroscopy studies have indicated that apoR2 undergoes a protein conformational change upon binding of metal (Tong et al. J. Am. Chem. Soc. 1996, 118, 2107-2108). On the basis of these observations, we propose a model for R2 metal incorporation that invokes an allosteric interaction between the two beta-peptides of R2. Upon binding the first equiv of metal to a beta-peptide (beta(I)), the aforementioned protein conformational change prevents metal binding in the adjacent beta-peptide (beta(II)) approximately 25 A away. Furthermore, we show that metal incorporation into beta(II) occurs only during the O(2) activation chemistry of the beta(I)-peptide. This is the first direct evidence of an allosteric interaction between the two beta-peptides of R2. Furthermore, this model can explain the generally observed low Fe occupancy of R2. We also demonstrate that metal uptake and this newly observed allosteric effect are buffer dependent. Higher levels of glycerol cause loss of the allosteric effect. Reductive cycling of samples in the presence of Mn(II) produced a novel mixed metal Fe(III)Mn(III)R2 species within the active site of R2. The magnitude of the exchange coupling (J) determined for both the Mn(2)(II)R2 and Fe(III)Mn(III)R2 species was determined to be -1.8 +/- 0.3 and -18 +/- 3 cm(-)(1), respectively. Quantitative spectral simulations for the Fe(III)Mn(III)R2 and mononuclear Mn(II)R2 species are provided. This work represents the first instance where both X- and Q-band simulations of perpendicular and parallel mode spectra were used to quantitatively predict the concentration of a protein bound mononuclear Mn(II) species.     

PELDOR study on the tyrosyl radicals in the R2 protein of mouse ribonucleotide reductase

Pulse electron-electron double resonance (PELDOR) has been employed to measure the distance between the putative tyrosyl radicals in the two halves of the R2 subunit from mouse ribonucleotide reductase. The results provide experimental evidence that the active, tyrosyl radical containing mouse R2 subunit forms a homodimeric form in solution. The distance between the two tyrosyl radicals present in the dimer was determined to be 3.25 +/- 0.05 nm.     

2,3-difluorotyrosine at position 356 of ribonucleotide reductase R2: a probe of long-range proton-coupled electron transfer

Escherichia coli class I ribonucleotide reductase catalyzes the conversion of ribonucleotides to deoxyribonucleotides and consists of two subunits: R1 and R2. R1 possesses the active site, while R2 harbors the essential diferric-tyrosyl radical (Y*) cofactor. The Y* on R2 is proposed to generate a transient thiyl radical on R1, 35 A distant, through amino acid radical intermediates. To study the putative long-range proton-coupled electron transfer (PCET), R2 (375 residues) was prepared semisynthetically using intein technology. Y356, a putative intermediate in the pathway, was replaced with 2,3-difluorotyrosine (F2Y, pKa = 7.8). pH rate profiles (pH 6.5-9.0) of wild-type and F2Y-R2 were very similar. Thus, a proton can be lost from the putative PCET pathway without affecting nucleotide reduction. The current model involving H* transfer is thus unlikely.     

Chelate control of diiron(I) dithiolates relevant to the [Fe-Fe]- hydrogenase active site

The reaction of Fe2(S2C2H4)(CO)6 with cis-Ph2PCH=CHPPh2 (dppv) yields Fe2(S2C2H4)(CO)4(dppv), 1(CO)4, wherein the dppv ligand is chelated to a single iron center. NMR analysis indicates that in 1(CO)4, the dppv ligand spans axial and basal coordination sites. In addition to the axial-basal isomer, the 1,3-propanedithiolate and azadithiolate derivatives exist as dibasal isomers. Density functional theory (DFT) calculations indicate that the axial-basal isomer is destabilized by nonbonding interactions between the dppv and the central NH or CH2 of the larger dithiolates. The Fe(CO)3 subunit in 1(CO)4 undergoes substitution with PMe3 and cyanide to afford 1(CO)3(PMe3) and (Et4N)[1(CN)(CO)3], respectively. Kinetic studies show that 1(CO)4 reacts faster with donor ligands than does its parent Fe2(S2C2H4)(CO)6. The rate of reaction of 1(CO)4 with PMe3 was first order in each reactant, k = 3.1 x 10(-4) M(-1) s(-1). The activation parameters for this substitution reaction, DeltaH = 5.8(5) kcal/mol and DeltaS = -48(2) cal/deg.mol, indicate an associative pathway. DFT calculations suggest that, relative to Fe2(S2C2H4)(CO)6, the enhanced electrophilicity of 1(CO)4 arises from the stabilization of a "rotated" transition state, which is favored by the unsymmetrically disposed donor ligands. Oxidation of MeCN solutions of 1(CO)3(PMe3) with Cp2FePF6 yielded [Fe2(S2C2H4)(mu-CO)(CO)2(dppv)(PMe3)(NCMe)](PF6)2. Reaction of this compound with PMe3 yielded [Fe2(S2C2H4)(mu-CO)(CO)(dppv)(PMe3)2(NCMe)](PF6)2.     

Water-dependent reactions of diiron(II) carboxylate complexes

Carboxylate-rich diiron(II) compounds with varying numbers of water ligands have been characterized, including the first complex with a {Fe2(mu-OH2)2(mu-O2CArTol)}3+ unit. The isolation of these complexes reveals how water can alter the structural properties of carboxylate-bridged diiron(II) core similar to those that occur in a variety of dioxygen-activating metalloenzyme cores. M?ssbauer and variable temperature, variable field magnetic susceptibility experiments indicate that the compound [Fe2(mu-OH2)2(mu-O2CAr4F-Ph)(O2CAr4F-Ph)3(THF)2(OH2)] has a high-spin diiron(II) core with little significant magnetic exchange coupling.     

High-valent diiron species generated from N-bridged diiron phthalocyanine and H(2)O(2)

N-bridged diiron tetra-tert-butylphthalocyanine activates H(2)O(2) to form anionic hydroperoxo complex [(Pc)Fe(IV)=N-Fe(III)(Pc)-OOH](-) prone to heterolytic cleavage of O-O bond with the release of OH(-) and formation of neutral diiron oxo phthalocyanine cation radical complex, PcFe(IV)=N-Fe(IV)(Pc(+)˙)=O. ESI-MS data showed stability of the Fe-N-Fe binuclear structure upon formation of this species, capable of oxidizing methane and benzene via O-atom transfer. The slow formation kinetics and the high reactivity preclude direct detection of this oxo complex by low temperature UV-vis spectroscopy. However, strong oxidizing properties and the results of EPR study support the formation of PcFe(IV)=N-Fe(IV)(Pc(+)˙)=O. Addition of H(2)O(2) at -80 °C led to the disappearance of iron EPR signal and to the appearance of the narrow signal at g = 2.001 consistent with the transient formation of PcFe(IV)=N-Fe(IV)(Pc(+)˙)=O. In the course of this study, another high valent diiron species was prepared in the solid state with 70% yield. The M?ssbauer spectrum shows two quadrupole doublets with δ(1) = -0.14 mm s(-1), ΔE(Q1) = 1.57 mm s(-1) and δ(2) = -0.10 mm s(-1), ΔE(Q2) = 2.03 mm s(-1), respectively. The negative δ values are consistent with formation of Fe(iv) states. Fe K-edge EXAFS spectroscopy reveals conservation of the diiron Fe-N-Fe core. In XANES, an intense 1s → 3d pre-edge feature at 7114.4 eV suggests formation of Fe(iv) species and attaching of one oxygen atom per two Fe atoms at the 1.90 ? distance. On the basis of M?ssbauer, EPR, EXAFS and XANES data this species was tentatively assigned as (Pc)Fe(IV)=N-Fe(IV)(Pc)-OH which could be formed from PcFe(IV)=N-Fe(IV)(Pc(+)˙)=O by hydrogen atom abstraction from a solvent molecule. Thus, despite unfavourable kinetics, we succeeded in the preparation of the first dirion(iv) phthalocyanine complex with oxygen ligand, generated in the (Pc)Fe(IV)=N-Fe(III)(Pc) - H(2)O(2) system capable of oxidizing methane.     

The pentacyanopyridine-4-aldoximeferrate(II) ion estimation of the ligand's coordination site

Summary Pyindine-4-aldoxime reacts with the aquopentacyanoferrate(II) ion by forming a complex compound of 1:1 molar ratio. ThepK _a value of the oxime group in the complex, the influence ofpH changes on its formation as well as IR data indicate the coordination of pyridine-4-aldoxime through the pyridine nitrogen.

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Das Pentacyanopyridin-4-aldoximferrat(II)-Ion. Bestimmung der Koordinationsstelle

Zusammenfassung Pyridin-4-aldoxim reagiert mit dem Aquopentacyanoferrat(II)-Ion und bildet einen Komplex mit dem Molarverhältnis 1:1. DerpK _a Wert der Oxim-Gruppe im Komplex, der Einfluß despH auf seine Bildung, wie auch die IR Daten weisen auf die Koordination des Pyridin-4-aldoxim über den Pyridin-Stickstoff hin.