Chemical Exchange at the Trinuclear Copper Center of Small Laccase from Streptomyces coelicolor.

The trinuclear copper center (TNC) of laccase reduces oxygen to water with very little overpotential. The arrangement of the coppers and ligands in the TNC is known to be from many crystal structures, yet information about possible dynamics of the ligands is absent. Here, we report dynamics at the TNC of small laccase from Streptomyces coelicolor using paramagnetic NMR and electron paramagnetic resonance spectroscopy. Fermi contact-shifted resonances tentatively assigned to histidine Hδ1 display a two-state chemical exchange with exchange rates in the order of 100 s-1. In the electron paramagnetic resonance spectra, at least two forms are observed with different gz-values. It is proposed that the exchange processes reflect the rotational motion of histidine imidazole rings that coordinate the coppers in the TNC.

Temperature-cycle electron paramagnetic resonance.

We report on a novel approach to the study of rates and short-lived intermediates of (bio)chemical reactions that involve paramagnetic species. Temperature-cycle Electron Paramagnetic Resonance (EPR) concerns the repeated heating of a reaction mixture in the cavity of an EPR spectrometer by pulsed irradiation with a near-infrared diode laser combined with intermittent characterization of the sample by 275 GHz EPR at a lower temperature at which the reaction does not proceed. The new technique is demonstrated for the reduction of TEMPOL with sodium dithionite in aqueous solution down to the sub-second time scale. We show that a single sample suffices to obtain a complete kinetic trace. Variation of the length and power of the laser pulse offers great flexibility as regards the time scale of the experiment and the temperature at which the reaction can be studied. For water/glycerol mixtures we introduce a simple way to obtain and load an unreacted sample into the spectrometer at low temperature.

Analysis of the EPR spectra of transferrin: the importance of a zero-field-splitting distribution and 4th-order terms.

Multi-frequency EPR spectroscopy can provide high-level structural information on high-spin Fe3+ sites in proteins and enzymes. Unfortunately, analysis of the EPR spectra of these spin systems is hindered by the presence of broad distributions in the zero-field-splitting (ZFS) parameters, which reflect conformational heterogeneity of the iron sites. We present the analysis of EPR spectra of high-spin Fe3+ bound to human serum transferrin. We apply a method termed the grid-of-errors to extract the distributions of the individual ZFS parameters from EPR spectra recorded in the high-field limit at a microwave frequency of 275 GHz. Study of a series of transferrin variants shows that the ZFS distributions are as characteristic of the structure of a high-spin Fe3+ site as the ZFS parameters themselves. Simulations based on the extracted ZFS distributions reproduce spectra recorded at 34 GHz (Q band) and 9.7 GHz (X band), including subtle variations that were previously difficult to quantify. The X-band spectrum of transferrin shows a characteristic double peak, which has puzzled researchers for decades. We show that the double peak is uniquely related to the term B4-3O4-3(S) in the spin Hamiltonian. Our method is generally applicable in the analysis of spectra that arise from a broad distribution of parameters.

Rapid Freeze-Quench EPR Spectroscopy: Improved Collection of Frozen Particles.

Rapid freeze-quench (RFQ) in combination with electron paramagnetic resonance (EPR) spectroscopy at X-band is a proven technique to trap and characterize paramagnetic intermediates of biochemical reactions. Preparation of suitable samples is still cumbersome, despite many attempts to remedy this problem, and limits the wide applicability of RFQ EPR. We present a method, which improves the collection of freeze-quench particles from isopentane and their packing in an EPR tube. The method is based on sucking the particle suspension into an EPR tube with a filter at the bottom. This procedure results in a significant reduction of the required volume of reactants, which allows the economical use of valuable reactants such as proteins. The approach also enables the successful collection of smaller frozen particles, which are generated at higher flow rates. The method provides for a reproducible, efficient and fast collection of the freeze-quench particles and can be easily adapted to RFQ EPR at higher microwave frequencies than X-band.

Exploring the Fe(III) binding sites of human serum transferrin with EPR at 275 GHz.

We report 275 GHz EPR spectra of human serum transferrin. At this high microwave frequency the zero-field splitting between the magnetic sublevels of the high-spin [Formula: see text] sites can be accurately determined. We find the zero-field splitting to be a sensitive probe of the structure of the transferrin iron-binding sites. Signals arising from iron bound to the transferrin N-lobe can clearly be distinguished from signals from iron bound to the C-lobe. Moreover, our spectra show that the structure of the iron site in the N-lobe is influenced by the presence and conformation of the C-lobe. The spectra of a series of N-lobe mutants altering the second-shell interaction of Arg124 with the synergistic anion carbonate reflect conformational changes induced at the iron site.

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.

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.

Stretched polyethylene films probed by single molecules.

Stretched films of low-density polyethylene (LDPE) doped with 2.3,8.9-dibenzanthanthrene (DBATT) were studied using polarization-selective single-molecule spectroscopy at 1.8 K. By measuring the in-plane component of the electronic transition-dipole moments of individual chromophores, the alignment of dopant molecules is determined without averaging. The distributions of chromophore orientations reveal the presence of two fractions of dopant molecules: those oriented along the stretching direction and randomly oriented molecules. With increasing drawing ratio of the polyethylene films, the ratio of oriented to randomly oriented guest molecules increases, whereas the extent of chromophore orientation, that is, the width of the orientation distribution, remains the same. The results are consistent with the interpretation that oriented chromophores reside on the surfaces of polyethylene crystals, instead of in the amorphous polyethylene regions. Guest molecules in stretched polyethylene are oriented due to the alignment of the crystallites on which they are adsorbed. As such, the shape and width of the distributions of chromophore orientations are determined by the interaction of guest molecules with the crystal surfaces.

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.

Configuration of spheroidene in the photosynthetic reaction center of Rhodobacter sphaeroides : a comparison of wild-type and reconstituted R26.

We compare the resonance Raman spectra acquired at two excitation wavelengths, 496.5 and 514.5 nm, of spheroidene in the wild-type reaction center of Rhodobacter sphaeroides and reconstituted into the reaction center of the Rhodobacter sphaeroides mutant R26. Our earlier work showed that the reconstituted R26 reaction center holds spheroidene in two configurations: 15,15'-cis and another configuration. Here we show that in the wild-type reaction center only 15,15'-cis spheroidene is present. In the resonance Raman spectra of the reconstituted R26 reaction centers, a transition is identified that arises exclusively from the second configuration. According to density-functional-theory calculations, this transition is specific for the 13,14-cis configuration.

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.

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.

Effective coupling of rapid freeze-quench to high-frequency electron paramagnetic resonance.

We report an easy, efficient and reproducible way to prepare Rapid-Freeze-Quench samples in sub-millimeter capillaries and load these into the probe head of a 275 GHz Electron Paramagnetic Resonance spectrometer. Kinetic data obtained for the binding reaction of azide to myoglobin demonstrate the feasibility of the method for high-frequency EPR. Experiments on the same samples at 9.5 GHz show that only a single series of Rapid-Freeze-Quench samples is required for studies at multiple microwave frequencies.

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.

Observer-selective double electron-electron-spin resonance, a pulse sequence to improve orientation selection.

By pulsed double electron-electron resonance (DEER), distances between spin labels in disordered systems up to 8 nm can be measured. In addition, the relative orientation of the interacting radicals can be determined, provided that the bandwidth of the pulses is sufficiently small. On the other hand, the bandwidth has to exceed the dipolar interaction considerably, because otherwise the DEER modulations become distorted and the modulation depth decreases, making distance determination impossible. Therefore, small bandwidths, i.e. long pulses, place a lower limit on the distance that can be determined. Two new pulse sequences, observer-selective DEER (os-DEER) and dead-time free os-DEER, are introduced that make it possible to use long observer pulses with bandwidths that are smaller than the dipolar interaction. The new pulse sequences do not suffer from the distortions caused by the limited bandwidth of the observer pulses, as demonstrated by measurements on a nitroxide biradical. With observer pulses of 140 ns, i.e., significantly longer than the 32 ns used in the conventional DEER sequence, a dipolar interaction of 7.8 MHz has been measured.

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.

Temperature Determination by EPR at 275 GHz and the Detection of Temperature Jumps in Aqueous Samples.

Second-moment analysis along two dimensions of continuous-wave EPR spectra of nitroxides enables EPR thermometry in a broad temperature range. Simulations show that the temperature can be derived in both the slow-motion and the fast-motion regime, which is experimentally verified at 275 GHz for H2O/glycerol (50/50% by volume) and pure water. We demonstrate that this tool allows the calibration of temperature jumps induced by infrared laser irradiation of a submicroliter sample in the single-mode cavity of a 275 GHz spectrometer, which prepares for kinetic studies of processes involving paramagnetic species.

Simulation of multi-frequency EPR spectra for a distribution of the zero-field splitting.

We present a numerical procedure called 'grid-of-errors' to extract the distribution of magnetic interactions from continuous-wave electron-paramagnetic-resonance (EPR) spectra at multiple microwave frequencies. The approach is based on the analysis of the lineshape of the spectra and explicitly worked out for high-spin systems for which the lineshape is determined by a distribution of the zero-field splitting. Initial principal values of the zero-field splitting tensor are obtained from the EPR spectrum at a microwave frequency in the high-field limit, and the initial distribution is taken Gaussian. Subsequently, the grid-of-errors procedure optimizes this distribution, without any restriction to its shape, taking into account spectra at various microwave frequencies. The numerical procedure is illustrated for the Fe(III)-EDTA complex. An optimized distribution of the zero-field splitting is obtained, which provides a proper description of the EPR spectra at 9.5, 34, 94, and 275 GHz. The proposed approach can be used as well for distributions of magnetic interactions other than the zero-field splitting.

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.

A Mononuclear Mn(II) Pseudoclathrochelate Complex Studied by Multi-Frequency Electron-Paramagnetic-Resonance Spectroscopy.

Knowledge of the correlation between structural and spectroscopic properties of transition-metal complexes is essential to deepen the understanding of their role in catalysis, molecular magnetism, and biological inorganic chemistry. It provides topological and, sometimes, functional insight with respect to the active site properties of metalloproteins. The electronic structure of a high-spin mononuclear Mn(II) pseudoclathrochelate complex has been investigated by electron-paramagnetic-resonance (EPR) spectroscopy at 9.5 and 275.7 GHz. A substantial, virtually axial zero-field splitting with D = -9.7 GHz (-0.32 cm(-1)) is found, which is the largest one reported to date for a Mn(II) complex with six nitrogen atoms in the first coordination sphere.