Magnetic anisotropy and structural flexibility in the field-induced single ion magnets [Co{(OPPh2)(EPPh2)N}2], E = S, Se, explored by experimental and computational methods.

During the last few years, a large number of mononuclear Co(II) complexes of various coordination geometries have been explored as potential single ion magnets (SIMs). In the work presented herein, the Co(II) S = 3/2 tetrahedral [Co{(OPPh2)(EPPh2)N}2], E = S, Se, complexes (abbreviated as CoO2E2), bearing chalcogenated mixed donor-atom imidodiphosphinato ligands, were studied by both experimental and computational techniques. Specifically, direct current (DC) magnetometry provided estimations of their zero-field splitting (zfs) axial (D) and rhombic (E) parameter values, which were more accurately determined by a combination of far-infrared magnetic spectroscopy and high-frequency and -field EPR spectroscopy studies. The latter combination of techniques was also implemented for the S = 3/2 tetrahedral [Co{(EPiPr2)2N}2], E = S, Se, complexes, confirming the previously determined magnitude of their zfs parameters. For both pairs of complexes (E = S, Se), it is concluded that the identity of the E donor atom does not significantly affect their zfs parameters. High-resolution multifrequency EPR studies of CoO2E2 provided evidence of multiple conformations, which are more clearly observed for CoO2Se2, in agreement with the structural disorder previously established for this complex by X-ray crystallography. The CoO2E2 complexes were shown to be field-induced SIMs, i.e., they exhibit slow relaxation of magnetization in the presence of an external DC magnetic field. Advanced quantum-chemical calculations on CoO2E2 provided additional insight into their electronic and structural properties.

Study of manganese binding to the ferroxidase centre of human H-type ferritin.

Ferritins are ubiquitous and conserved proteins endowed with enzymatic ferroxidase activity, that oxidize Fe(II) ions at the dimetal ferroxidase centre to form a mineralized Fe(III) oxide core deposited within the apo-protein shell. Herein, the in vitro formation of a heterodimetal cofactor constituted by Fe and Mn ions has been investigated in human H ferritin (hHFt). Namely, Mn and Fe binding at the hHFt ferroxidase centre and its effects on Fe(II) oxidation have been investigated by UV-Vis ferroxidation kinetics, fluorimetric titrations, multifrequency EPR, and preliminary Mössbauer spectroscopy. Our results show that in hHFt, both Fe(II) and Mn(II) bind the ferroxidase centre forming a Fe-Mn cofactor. Moreover, molecular oxygen seems to favour Mn(II) binding and increases the ferroxidation activity of the Mn-loaded protein. The data suggest that Mn influences the Fe binding and the efficiency of the ferroxidation reaction. The higher efficiency of the Mn-Fe heterometallic centre may have a physiological relevance in specific cell types (i.e. glia cells), where the concentration of Mn is the same order of magnitude as iron.

Magnetic Anisotropy of Tetrahedral CoII Single-Ion Magnets: Solid-State Effects.

This study reports the static and dynamic magnetic characterization of two mononuclear tetrahedral CoII complexes, [Co{iPr2P(E)NP(E)iPr2}2], where E = S (CoS4) and Se (CoSe4), which behave as single-ion magnets (SIMs). Low-temperature (15 K) single-crystal X-ray diffraction studies point out that the two complexes exhibit similar structural features in their first coordination sphere, but a disordered peripheral iPr group is observed only in CoS4. Although the latter complex crystallizes in an axial space group, the observed structural disorder leads to larger transverse magnetic anisotropy for the majority of the molecules compared to CoSe4, as confirmed by electron paramagnetic resonance spectroscopy. Static magnetic characterization indicates that both CoS4 and CoSe4 show easy-axis anisotropy, with comparable D values (∼-30 cm-1). Moreover, alternating-current susceptibility measurements on these CoII complexes, magnetically diluted in their isostructural ZnII analogues, highlight the role of dipolar magnetic coupling in the mechanism of magnetization reversal. In addition, our findings suggest that, despite their similar anisotropic features, CoS4 and CoSe4 relax magnetically via different processes. This work provides experimental evidence that solid-state effects may affect the magnetic behavior of SIMs.

The type 1 copper site of pseudoazurin: axial and rhombic.

We report on a high-frequency electron-paramagnetic-resonance study of the type 1 copper site of pseudoazurin. The spectra fully resolve the contribution of a nearly axial spectrum besides the rhombic spectrum, which unequivocally proves the existence of two conformations of the copper site. Pseudoazurins have been considered from Achromobacter cycloclastes including eight mutants and from Alcaligenes faecalis. The two conformations are virtually the same for all pseudoazurins, but the rhombic/axial population varies largely, between 91/9 and 33/67. These observations are discussed in relation to optical absorption spectra and X-ray diffraction structures. A similar observation for fern plastocyanin from Dryopteris crassirhizoma suggests that dual conformations of type 1 copper sites are more common.

Copper sulfide nanocrystals with tunable composition by reduction of covellite nanocrystals with Cu+ ions.

Platelet-shaped copper sulfide nanocrystals (NCs) with tunable Cu stoichiometry were prepared from Cu-rich covellite (Cu1.1S) nanoplates through their reaction with a Cu(I) complex ([Cu(CH3CN)4]PF6) at room temperature. Starting from a common sample, by this approach it is possible to access a range of compositions in these NCs, varying from Cu1.1S up to Cu2S, each characterized by a different optical response: from the metallic covellite, with a high density of free carriers and strong localized surface plasmon resonance (LSPR), up to Cu2S NCs with no LSPR. In all these NCs the valency of Cu in the lattice stays always close to +1, while the average -1 valency of S in covellite gradually evolves to -2 with increasing Cu content; i.e., sulfur is progressively reduced. The addition of copper to the starting covellite NCs is similar to the intercalation of metal species in layered transition metal dichalcogenides (TMDCs); i.e., the chalcogen-chalcogen bonds holding the layers are progressively broken to make room for the intercalated metals, while their overall anion sublattice does not change much. However, differently from the TMDCs, the intercalation in covellite NCs is sustained by a change in the redox state of the anion framework. Furthermore, the amount of Cu incorporated in the NCs upon reaction is associated with the formation of an equimolar amount of Cu(II) species in solution. Therefore, the reaction scheme can be written as: Cu1.1S + 2γCu(I) → Cu1.1+γS + γCu(II).

Involvement of Tyr108 in the enzyme mechanism of the small laccase from Streptomyces coelicolor.

The enzyme mechanism of the multicopper oxidase (MCO) SLAC from Streptomyces coelicolor was investigated by structural (XRD), spectroscopic (optical, EPR), and kinetics (stopped-flow) experiments on variants in which residue Tyr108 had been replaced by Phe or Ala through site-directed mutagenesis. Contrary to the more common three-domain MCOs, a tyrosine in the two-domain SLAC is found to participate in the enzyme mechanism by providing an electron during oxygen reduction, giving rise to the temporary appearance of a tyrosyl radical. The relatively low k(cat)/K(M) of SLAC and the involvement of Y108 in the enzyme mechanism may reflect an adaptation to a milieu in which there is an imbalance between the available reducing and oxidizing co-substrates. The purported evolutionary relationship between the two-domain MCOs and human ceruloplasmin appears to extend not only to the 3D structure and the mode of binding of the Cu's in the trinuclear center, as noted before, but also to the enzyme mechanism.

High-frequency EPR study of the high-spin FeII complex Fe[(SPPh2)2N]2.

We report continuous-wave electron-paramagnetic-resonance (EPR) spectra of the high-spin Fe(II) complex Fe[(SPPh(2))(2)N](2) at 275.7 GHz, 94.1 GHz and 9.5 GHz. Combined analysis of these EPR spectra shows that the complex occurs in multiple conformations. For two main conformations the spin-Hamiltonian parameters, which reflect the electronic structure of the complex, are accurately determined: (1) D=9.17 cm(-1) (275 GHz), E/D=0.021 and (2) D=8.87 cm(-1) (266 GHz), E/D=0.052. The EPR spectra obtained at 275.7 GHz on single crystals of the complex are essential for the analysis and in addition they reveal that the two main conformations occur at two magnetically distinguishable sites.

A comment on the pseudo-nuclear Zeeman effect.

For high-spin systems whose magnetic sublevels are arranged in doublets at zero field, the electron-paramagnetic-resonance (EPR) spectra are commonly described by an effective spin Hamiltonian. We show that also in this approach, if the mixing of the electron spin states by the hyperfine interaction is negligible, a proper description of electron-nuclear double resonance (ENDOR) spectra can be obtained using a nuclear spin Hamiltonian in which the electron spin angular momentum operator is replaced by its expectation value. Appropriate values of this expectation value can be obtained from a wave function correct to first-order in the electron Zeeman interaction. In terms of perturbation theory, such a description is more logical than the conventional practice based on the inclusion of a second-order cross term, the so-called pseudo-nuclear Zeeman effect, which involves both the electron Zeeman interaction and the hyperfine interaction. We illustrate our analysis with calculations of the expectation value of the electron spin angular momentum and of the energies of the hyperfine levels for a high-spin cobalt complex, which we studied by EPR and ENDOR recently.

Theoretical analysis of the spin Hamiltonian parameters in Co(II)S4 complexes, using density functional theory and correlated ab initio methods.

A systematic Density Functional Theory (DFT) and multiconfigurational ab initio computational analysis of the Spin Hamiltonian (SH) parameters of tetracoordinate S = 3/2 Co((II))S(4)-containing complexes has been performed. The complexes under study bear either arylthiolato, ArS(-), or dithioimidodiphosphinato, [R(2)P(S)NP(S)R'(2)](-) ligands. These complexes were chosen because accurate structural and spectroscopic data are available, including extensive Electron Paramagnetic Resonance (EPR)/Electron Nuclear Double Resonance (ENDOR) studies. For comparison purposes, the [Co(PPh(3))(2)Cl(2)] complex, which was thoroughly studied in the past by High-Field and Frequency EPR and Variable Temperature, Variable Field Magnetic Circular Dichroism (MCD) spectroscopies, was included in the studied set. The magnitude of the computed axial zero-field splitting parameter D (ZFS), of the Co((II))S(4) systems, was found to be within ~10% of the experimental values, provided that the property calculation is taken beyond the accuracy obtained with a second-order treatment of the spin-orbit coupling interaction. This is achieved by quasi degenerate perturbation theory (QDPT), in conjunction with complete active space configuration interaction (CAS-CI). The accuracy was increased upon recovering dynamic correlation with multiconfigurational ab initio methods. Specifically, spectroscopy oriented configuration interaction (SORCI), and difference dedicated configuration interaction (DDCI) were employed for the calculation of the D-tensor. The sign and magnitude of parameter D was analyzed in the framework of Ligand Field Theory, to reveal the differences in the electronic structures of the investigated Co((II))S(4) systems. For the axial complexes, accurate effective g'-tensors were obtained in the QDPT studies. These provide a diagnostic tool for the adopted ground state configuration (±3/2 or ±1/2) and are hence indicative of the sign of D. On the other hand, for the rhombic complexes, the determination of the sign of D required the SH parameters to be derived along suitably constructed symmetry interconversion pathways. This procedure, which introduces a dynamic perspective into the theoretical investigation, helped to shed some light on unresolved issues of the corresponding experimental studies. The metal hyperfine and ligand super-hyperfine A-tensors of the C(2) [Co{(SPPh(2))(SP(i)Pr(2))N}(2)] complex were estimated by DFT calculations. The theoretical data were shown to be in good agreement with the available experimental data. Decomposition of the metal A-tensor into individual contributions revealed that, despite the large ZFS, the observed significant anisotropy should be largely attributed to spin-dipolar contributions. The analysis of both, metal and ligand A-tensors, is consistent with a highly covalent character of the Co-S bonds.

A Proton ENDOR Study of Azurin.

As part of our ongoing project that aims at the optimum characterization of the electronic structure of the blue-copper site of azurin from Pseudomonas aeruginosa, we present the complete hyperfine tensors of the protons bound to the Cbeta atom of the copper-bound cysteine 112. These tensors have been obtained from a 95 GHz pulsed electron-nuclear double resonance study of a single crystal of the protein.

A multifrequency high-field electron paramagnetic resonance study of Co(II)S(4) coordination.

Advanced electron paramagnetic resonance (EPR) methods have been employed in the study of two high-spin cobalt(II) complexes, Co[(SPPh(2))(2)N](2) (Co(Ph,Ph)L(2)) and Co[(SPPh(2))(SP(i)Pr(2))N](2) (Co(iPr,Ph)L(2)), in which the bidentate disulfidoimidodiphosphinato ligands make up for a pseudotetrahedral sulfur coordination of the transition metal. The CoS(4) core in the two complexes has slightly different structure, owing to the different peripheral groups (phenyl or isopropyl) bound to the phosphorus atoms. To determine the zero-field splitting, notoriously difficult for high-spin cobalt(II), the two complexes required different approaches. For Co(Ph,Ph)L(2), the study of the X-band EPR spectrum of a single crystal as a function of temperature revealed a nearly axial character of the zero-field splitting (ZFS; E/D approximately -0.05). For Co(iPr,Ph)L(2), the combination of the EPR spectra at 9, 95, and 275 GHz revealed a rhombic character of the ZFS (E/D approximately -0.33). The energy difference between the Kramers doublets in Co(Ph,Ph)L(2) and Co(iPr,Ph)L(2) amounts to 24 cm(-1) and 30 cm(-1), respectively. From the X-band EPR spectra of diamagnetically diluted single crystals at fields up to 2.5 T for Co(Ph,Ph)L(2) and 0.5 T for Co(iPr,Ph)L(2), the effective g tensors and cobalt hyperfine tensors have been determined, including the direction of the principal axes in the cobalt sites. The values of the EPR observables are discussed in relation to the structural characteristics of the first (CoS(4)) and second coordination sphere in the complexes.

Identification of a radical intermediate in the enzymatic reduction of oxygen by a small laccase.

The enzyme mechanism of the Cu-containing small laccase (SLAC) from Streptomyces coelicolor has been investigated by optical and electron paramagnetic resonance spectroscopy. A new intermediate was identified after the reaction of molecular oxygen with the reduced trinuclear site of the type-1-depleted (T1D) form of the enzyme. It has the fingerprint of a biradical with a triplet ground state. One of the spins resides on a Cu in the trinuclear site, tentatively identified as the type-2 site, while the other spin derives from a protein-based radical. The latter is tentatively identified as a tyrosyl radical on the basis of the similarity of the optical characteristics with those observed for a Cu tyrosyl radical pair. The spin-spin distance was found to be 5.0 +/- 0.2 A.

A W-band pulsed EPR/ENDOR study of Co(II)S(4) coordination in the Co[(SPPh(2))(SP(i)Pr(2))N](2) complex.

Spin-echo detection at 95 GHz enables an electron-paramagnetic-resonance study of a cobalt complex with a bio-mimetic coordination of the transition metal by four sulfur atoms. A magnetically diluted single crystal of the complex has been investigated in great detail. Electron-nuclear double-resonance signals were observed of ligand nuclei and complete hyperfine tensors of the distinct phosphorus nuclei were derived, assigned and discussed.

A modification of the commercial ESR900 cryostat to enable three-dimensional electron-paramagnetic-resonance studies of crystals.

Complete orientation studies of X-band electron-paramagnetic-resonance spectra of crystals largely benefit from the possibility to measure the spectrum for any orientation of the magnetic field with respect to the crystal without the need to remount the crystal. We report on a modification of a commercial cryostat to allow such experiments down to liquid helium temperatures and demonstrate its performance.

Acid-induced unfolding of myoglobin triggered by a laser pH jump method.

Using 1-(2-nitrophenyl)ethyl sulfate (caged sulfate) as a photoactivatable caged proton, we could induce complete acid unfolding of myoglobin with a single nanosecond laser pulse. This was possible because of the high ( approximately mM) concentration of protons released by the photolabile compound. The ability of the compound to produce a large pH jump arises because the other photoproducts (2-nitrosoacetophenone and sulfate ion) do not buffer the released protons. The complete time course of the unfolding kinetics, spanning a range from milliseconds to several seconds, could be accurately reproduced by monitoring absorbance changes in the visible spectrum at 633 nm.

Geminate rebinding in R-state hemoglobin: kinetic and computational evidence for multiple hydrophobic pockets.

Biphasic geminate rebinding of CO to myoglobin upon flash photolysis has been associated to ligand distribution in hydrophobic cavities, structurally detected by time-resolved crystallography, xenon occupancy, and molecular simulations. We show that the time course of CO rebinding to human hemoglobin also exhibits a biphasic geminate rebinding when the protein is entrapped in wet nanoporous silica gel. A simple branched kinetic scheme, involving the bound state A, the primary docking site C, and a secondary binding site B was used to calculate the microscopic rates and the time-dependent population of the intermediate species. The activation enthalpies of the associated transitions were determined in the absence and presence of 80% glycerol. Potential hydrophobic docking cavities within the alpha and beta chains of hemoglobin were identified by computational modeling using xenon as a probe. A hydrophobic pocket on the distal side of the heme, corresponding to Xe4 in Mb, and a nearby site that does not have a correspondence in Mb were detected. Neither potential xenon sites on the proximal side nor a migration channel from the distal to proximal site was located. The small enthalpic barriers between states B and C are in very good agreement with the location of the xenon sites on the distal side. Furthermore, the connection between the two xenon sites is relatively open, explaining why the decreased mobility of the protein with viscosity only slightly perturbs the energetics of ligand migration between the two sites.

Light-induced protein-matrix uncoupling and protein relaxation in dry samples of trehalose-coated MbCO at room temperature.

In humid samples of trehalose-coated carboxy-myoglobin (MbCO), thermally driven conformational relaxation takes place after photodissociation of the carbon monoxide (CO) molecule at room temperature. In such samples, because of the extreme viscosity of the external matrix, photodissociated CO cannot diffuse out of the protein and explores the whole (proximal and distal side) heme pocket, experiencing averaged protein heme pocket structures, as a result of the presence of Brownian motions. At variance, in very dry samples, a lower portion of the photodissociated CO diffuses from the distal to the proximal heme pocket side probing in nonaveraged structures. We revisit here the flash photolysis data by Librizzi et al. (2002) and report on new, room temperature experiments in MbCO-trehalose samples, shortly illuminated prior the laser pulse. In dry samples, pre-illumination increased the diffusion of CO from the distal to the proximal heme pocket side, which resulted in less structure than in non-pre-illuminated samples. Such an effect, which is absent in humid samples, stems from a decoupling of the protein internal degrees of freedom from those of the external water-sugar matrix. We suggest that such a decoupling can be brought about by the continuous attempts performed by the protein during pre-illumination to undergo relaxation toward the photodissociated deoxy state. This, in turn, causes a collapse in the hydrogen bond network, which connects the protein surface to the water-sugar matrix, as reported by Cottone et al. (2002) and Giuffrida et al. (2003). In the conclusion section, we discuss the possible involvement of the processes invoked to rationalize the present data, in the function of macromolecules and interactions in living cells.

Kinetics of proton release after flash photolysis of 1-(2-nitrophenyl)ethyl sulfate (caged sulfate) in aqueous solution.

The kinetics of proton release after laser photolysis of 1-(2-nitrophenyl)ethyl sulfate (caged sulfate) have been characterized by time-resolved absorbance and photoacoustic methods. The absorbance at approximately 400 nm is observed to rise with a biphasic behavior in which a prompt component (formation of the nitronic acid) is followed by a slower (tau approximately 63 +/- 6 ns) phase (deprotonation of the nitronic acid). The decay of this intermediate occurs with a lifetime which is affected by the pH of the solution and the laser pulse energy. In buffered aqueous solution at pH 7, 20 degrees C the aci-nitro decay rate is 18 +/- 4 s(-1). Protons are released to the solution with rate (1.58 +/- 0.09) x 10(7) s(-1) at neutral pH from the nitronic acid intermediate. From the numerical analysis of the protonation kinetics of suitable pH indicators, we could estimate the pK(a) of the nitronic acid as 3.69 +/- 0.05. At acidic pH, a substantial fraction of the aci-nitro intermediate is in the protonated form and this leads to a biphasic release of protons, with the slower phase being characterized by an apparent rate constant strongly dependent on the pH. The strongly acidic character of the final photoproduct (sulfate ion) means that there is negligible buffering of photoreleased protons.

Evidence for two geminate rebinding states following laser photolysis of R state hemoglobin encapsulated in wet silica gels.

In this letter we report the first experimental evidence for CO rebinding to human hemoglobin from multiple geminate states. The analysis of the rebinding kinetics using a maximum entropy method allowed the identification of two distinct rebinding states within the protein matrix, which become populated under conditions of increased viscosity in a silica gel at high glycerol concentration. Our findings suggest the presence of at least two distinct docking sites for the photolyzed ligand. Assuming a minimal four-state model, we estimate the microscopic rates and the activation energies for the elementary processes.

Determination of microscopic rate constants for CO binding and migration in myoglobin encapsulated in silica gels.

CO rebinding kinetics after nanosecond photolysis of myoglobin encapsulated in wet silica gels exhibits an enhanced geminate phase that allows the determination of the microscopic rate constants and the activation barriers for distinct ligand docking sites inside the protein matrix. Using a maximum entropy method, we demonstrate that the geminate phase can be well-described by a biphasic lifetime distribution, reflecting rebinding from the distal and proximal sites. Microscopic rates and activation barriers were estimated using a four-state model.