Spin-density distribution in the copper site of azurin.

A 95 GHz pulsed deuterium ENDOR study has been performed on single crystals of azurin from Pseudomonas aeruginosa selectively deuterated at the C(beta) position of the copper-coordinating cysteine 112. Complete hyperfine tensors of the two deuterium atoms have been obtained, which reveal identical isotropic parts. Analysis of the hyperfine tensors provides insight into the spin-density delocalization over the cysteine ligand. Approximately 45 % of the spin density in the paramagnetic site can be attributed to copper and 30 % to sulfur.     

Comparative density-functional study of the electron paramagnetic resonance parameters of amavadin

Electronic g tensors and hyperfine coupling tensors have been calculated for amavadin, an unusual eight-coordinate vanadium(IV) complex isolated from Amanita muscaria mushrooms. Different density-functional methods have been compared, ranging from local via gradient-corrected to hybrid functionals with a variable Hartree-Fock exchange admixture. For both electron paramagnetic resonance (EPR) properties, hybrid functionals with an appreciable exact-exchange admixture provide the closest agreement with experimental data. Second-order spin-orbit corrections provide non-negligible contributions to the 51V hyperfine tensor. The orientation of g and A tensors relative to each other also depends on spin-orbit corrections to the A tensor. A rationalization for the close resemblance of the EPR parameters of amavadin to those of the structurally rather different vanadyl complexes is provided, based on the nature of the relevant frontier orbitals.     

Computational studies of electron paramagnetic resonance parameters for paramagnetic molybdenum complexes. 1. Method validation on small and medium-sized systems

A variety of density functional methods have been evaluated in the computation of electronic g-tensors and molybdenum hyperfine couplings for systems ranging from the Mo atom through MoIIIN, [MoVOCl4]-, and [MoVOF5]2- to two larger MoV complexes MoXLCl2 (X=O, S; L=tris(3,5-dimethylpyrazolyl)hydroborate anion). In particular, the influence of the molybdenum basis set and of various exchange-correlation functionals with variable admixtures of Hartree-Fock exchange on the computed EPR parameters have been evaluated in detail. Careful basis-set studies have provided a moderate-sized 12s6p5d all-electron basis on molybdenum that gives hyperfine tensors in excellent agreement with much larger basis sets and that will be useful for calculations on larger systems. The best agreement with experimental data for both hyperfine and g-tensors is obtained with hybrid functionals containing approximately 30-40% Hartree-Fock exchange. Only for MoSLCl2 does increasing spin contamination with increasing exact-exchange admixture restrict the achievable computational accuracy. In all cases, spin-orbit corrections to the hyperfine tensors are sizable and have to be included in accurate calculations. Scalar relativistic effects enhance the isotropic Mo hyperfine coupling by approximately 15-20%. Two-component g-tensor calculations with variational inclusion of spin-orbit coupling show that the Deltag parallel components in [MoVOCl4]- and [MoVOF5]2- depend on higher-order spin-orbit contributions and are thus described insufficiently by the usual second-order perturbation approaches. Computed orientations of g- and hyperfine tensors relative to each other and to the molecular framework for the MoXLCl2 complexes provide good agreement between theory and single-crystal electron paramagnetic resonance experiments. In these cases, the hyperfine tensor orientations are influenced only slightly by spin-orbit effects.     

Computational studies of EPR parameters for paramagnetic molybdenum complexes. II. Larger MoV systems relevant to molybdenum enzymes

The careful validation of modern density functional methods for the computation of electron paramagnetic resonance (EPR) parameters in molybdenum complexes has been extended to a number of low-symmetry MoV systems that model molybdoenzyme active sites. Both g and hyperfine tensors tend to be reproduced best by hybrid density functionals with about 30-40% exact-exchange admixture, with no particular spin contamination problems encountered. Spin-orbit corrections to hyperfine tensors are mandatory for quantitative and, in some cases, even for qualitative agreement. The g11 (g||) component of the g tensor tends to come out too positive when spin-orbit coupling is included only to leading order in perturbation theory. Compared to single-crystal experiments, the calculations reproduce both g- and hyperfine-tensor orientations well, both relative to each other and to the molecular framework. This is significant, as simulations of the EPR spectra of natural-abundance frozen-solution samples frequently do not allow a reliable determination of the hyperfine tensors. These may now be extracted based on the quantum-chemically calculated parameters. In a number of cases, revised simulations of the experimental spectra have brought theory and experiment into substantially improved agreement. Systems with two terminal oxo ligands, and to some extent with an oxo and a sulfido ligand, have been confirmed to exhibit particularly large negative Deltag33 shifts and thus large g anisotropies. This is discussed in the context of the experimental data for xanthine oxidase.     

Understanding the electon paramagnetic resonance parameters of protein-bound glycyl radicals

The number of enzymes that require a glycyl-based radical for their function is growing. Here, we provide systematic quantum-chemical studies of spin-density distributions, electronic g-tensors, and hyperfine couplings of various models of protein-bound glycyl radicals. Similarly to what is found in a companion paper on N-acetylglycyl, the small g-anisotropy for this delocalized, unsymmetrical system presents appreciable challenges to state-of-the-art computational methodology. This pertains to the quality of structure optimization, as well as to the choice of the spin-orbit Hamiltonian and the gauge origin of the magnetic vector potential. Environmental effects due to hydrogen bonding are complicated and depend in a subtle fashion on the different intramolecular hydrogen bonding for different conformations of the radical. Indeed, the conformation has the largest overall effect on the computed g-tensors (less so on the hyperfine tensors). This is discussed in the context of different g-tensors obtained by recent high-field electron paramagnetic resonance (EPR) measurements for three different enzymes. On the basis of results of a parallel calibration study for N-acetylglycyl, it is suggested that the glycyl radical observed for E. coli anaerobic RNR may have a fully extended conformation, which differs from those of the corresponding radicals in pyruvate formate-lyase or benzylsuccinate synthase.     

Spin delocalization over type zero copper

Hard-ligand, high-potential copper sites have been characterized in double mutants of Pseudomonas aeruginosa azurin (C112D/M121X (X = L, F, I)). These sites feature a small A(zz)(Cu) splitting in the EPR spectrum together with enhanced electron transfer activity. Due to these unique properties, these constructs have been called "type zero" copper sites. In contrast, the single mutant, C112D, features a large A(zz)(Cu) value characteristic of the typical type 2 Cu(II). In general, A(zz)(Cu) comprises contributions from Fermi contact, spin dipolar, and orbital dipolar terms. In order to understand the origin of the low A(zz)(Cu) value of type zero Cu(II), we explored in detail its degree of covalency, as manifested by spin delocalization over its ligands, which affects A(zz)(Cu) through the Fermi contact and spin dipolar contributions. This was achieved by the application of several complementary EPR hyperfine spectroscopic techniques at X- and W-band (～9.5 and 95 GHz, respectively) frequencies to map the ligand hyperfine couplings. Our results show that spin delocalization over the ligands in type zero Cu(II) is different from that of type 2 Cu(II) in the single C112D mutant. The (14)N hyperfine couplings of the coordinated histidine nitrogens are smaller by about 25-40%, whereas that of the (13)C carboxylate of D112 is about 50% larger. From this comparison, we concluded that the spin delocalization of type zero copper over its ligands is not dramatically larger than in type 2 C112D. Therefore, the reduced A(zz)(Cu) value of type zero Cu(II) is largely attributable to an increased orbital dipolar contribution that is related to its larger g(zz) value, as a consequence of the distorted tetrahedral geometry. The increased spin delocalization over the D112 carboxylate in type zero mutants compared to type 2 C112D suggests that electron transfer paths involving this residue are enhanced.     

Probing the role of axial methionine in the blue copper center of azurin with unnatural amino acids

Expressed protein ligation was used to replace the axial methionine of the blue copper protein azurin from Pseudomonas aeruginosa with unnatural amino acids. The highly conserved methionine121 residue was replaced with the isostructural amino acids norleucine (Nle) and selenomethionine (SeM). The UV-visible absorption, X- and Q-band EPR, and Cu EXAFS spectra of the variants are slightly perturbed from WT. All variants have a predominant S(Cys) to Cu(II) charge transfer band around 625 nm and narrow EPR hyperfine splittings. The Se EXAFS of the M121SeM variant is also reported. In contrast to the small spectral changes, the reduction potentials of M121SeM, M121Leu, and M121Nle are 25, 135, and 140 mV, respectively, higher than that of WT azurin. The use of unnatural amino acids allowed deconvolution of different factors affecting the reduction potentials of the blue copper center. A careful analysis of the WT azurin and its variants obtained in this work showed the large reduction potential variation was linearly correlated with the hydrophobicity of the axial ligand side chains. Therefore, hydrophobicity is the dominant factor in tuning the reduction potentials of blue copper centers by axial ligands.     

Theoretical study of the external heavy atom effect on phosphorescence of free-base porphin molecule

The radiative lifetime of phosphorescence of free-base porphin (H2P) molecule and its complexes with noble-gas atoms are calculated by time-dependent density functions theory (TD DFT) with quadratic response functions for account of spin-orbit coupling and electric dipole activity. The complexes with Ne, Ar, Kr, and Xe are used to simulate the external heavy atom (EHA) effect on phosphorescence of the H2P molecule in the corresponding noble gas matrices. The B3LYP functional and small basis set (3-21G) are used throughout the study and comparison of all complexes but other basis sets are also utilized to support the chosen approach. A slow radiative rate constant of free-base porphin phosphorescence (about 10(-3) s(-1)) is obtained with all basis sets being in the order of magnitude agreement with experimental estimations. A strong enhancement of the H2P phosphorescence rate (by 360 times) is calculated for Xe complex; while for Ne, Ar, and Kr complexes, the enhancement is equal to 1.1, 1.3, and 10.3 times, respectively. In these complexes, the noble gas atom is disposed at 3.6 A above the center of the porphin ring. In spite of shortcomings of the chosen simple model, the TD DFT calculations explain the most important features of the EHA effect on phosphorescence of free-base porphin. Calculations of the hyperfine coupling tensors for all magnetic nuclei in the lowest triplet state of H2P molecule and its complexes with noble-gas atoms indicate an appreciable penetration of the spin density to the EHA region. This can be connected with the enhancement of spin-orbit coupling in the H2P molecule.     

NMR hyperfine shifts in blue copper proteins: a quantum chemical investigation

We present the results of the first quantum chemical investigations of 1H NMR hyperfine shifts in the blue copper proteins (BCPs): amicyanin, azurin, pseudoazurin, plastocyanin, stellacyanin, and rusticyanin. We find that very large structural models that incorporate extensive hydrogen bond networks, as well as geometry optimization, are required to reproduce the experimental NMR hyperfine shift results, the best theory vs experiment predictions having R2 = 0.94, a slope = 1.01, and a SD = 40.5 ppm (or approximately 4.7% of the overall approximately 860 ppm shift range). We also find interesting correlations between the hyperfine shifts and the bond and ring critical point properties computed using atoms-in-molecules theory, in addition to finding that hyperfine shifts can be well-predicted by using an empirical model, based on the geometry-optimized structures, which in the future should be of use in structure refinement.     

Structure and electron paramagnetic resonance parameters of the manganese site of concanavalin A studied by density functional methods

The EPR parameters of the manganese site in the saccharide-binding protein concanavalin A have been studied by density functional methods, with an emphasis on metal (⁵⁵Mn) and ligand (¹H and ¹⁷O) hyperfine couplings, in comparison with high-field EPR and ENDOR data. Results for gradient-corrected and hybrid functionals with different exact-exchange admixture have been compared with experiment for the ⁵⁵Mn and the ¹H ligand hyperfine coupling and have been predicted for ¹⁷O hyperfine coupling based on comparison with experiment for the related [Mn(H₂O)₆]²⁺. Appreciable exact-exchange admixture in the hybrid functional is needed to obtain an adequate spin-density distribution and thus near-quantitative agreement with experimental EPR parameters. The common use of experimental proton hyperfine coupling tensors together with the point-dipole approximation for determination of bond lengths is evaluated by explicit calculations.     

Characterisation of [Cu4S], the catalytic site in nitrous oxide reductase, by EPR spectroscopy

The enzyme nitrous oxide reductase (N(2)OR) has a unique tetranuclear copper centre [Cu(4)S], called Cu(Z), at the catalytic site for the two-electron reduction of N(2)O to N(2). The X- and Q-band EPR spectra have been recorded from two forms of the catalytic site of the enzyme N(2)OR from Paracoccus pantotrophus, namely, a form prepared anaerobically, Cu(Z), that undergoes a one-electron redox cycle and Cu(Z)*, prepared aerobically, which cannot be redox cycled. The spectra of both species are axial with that of Cu(Z) showing a rich hyperfine splitting in the g||-region at X-band. DFT calculations were performed to gain insight into the electronic configuration and ground-state properties of Cu(Z) and to calculate EPR parameters. The results for the oxidation state [Cu(+1)(3)Cu(+2)(1)S](3+) are in good agreement with values obtained from the fitting of experimental spectra, confirming the absolute oxidation state of Cu(Z). The unpaired spin density in this configuration is delocalised over four copper ions, thus, Cu(I) 20.1%, Cu(II) 9.5%, Cu(III) 4.8% and Cu(IV) 9.2%, the mu(4)-sulfide ion and oxygen ligand. The three copper ions carrying the highest spin density plus the sulfide ion lie approximately in the same plane while the fourth copper ion is perpendicular to this plane and carries only 4.8% spin density. It is suggested that the atoms in this plane represent the catalytic core of Cu(Z), allowing electron redistribution within the plane during interaction with the substrate, N(2)O.     

EPR-ENDOR of the Cu(I)NO complex of nitrite reductase

With limited reductant and nitrite under anaerobic conditions, copper-containing nitrite reductase (NiR) of Rhodobacter sphaeroides yielded endogenous NO and the Cu(I)NO derivative of NiR. (14)N- and (15)N-nitrite substrates gave rise to characteristic (14)NO and (15)NO EPR hyperfine features indicating NO involvement, and enrichment of NiR with (63)Cu isotope caused an EPR line shape change showing copper involvement. A markedly similar Cu(I)NONiR complex was made by anaerobically adding a little endogenous NO gas to reduced protein and immediately freezing. The Cu(I)NONiR signal accounted for 60-90% of the integrated EPR intensity formerly associated with the Type 2 catalytic copper. Analysis of NO and Cu hyperfine couplings and comparison to couplings of inorganic Cu(I)NO model systems indicated approximately 50% spin on the N of NO and approximately 17% spin on Cu. ENDOR revealed weak nitrogen hyperfine coupling to one or more likely histidine ligands of copper. Although previous crystallography of the conservative I289V mutant had shown no structural change beyond the 289 position, this mutation, which eliminates the Cdelta1 methyl of I289, caused the Cu(I)NONiR EPR spectrum to change and proton ENDOR features to be significantly altered. The proton hyperfine coupling that was significantly altered was consistent with a dipolar interaction between the Cdelta1 protons of I289 and electron spin on the NO, where the NO would be located 3.0-3.7 A from these protons. Such a distance positions the NO of Cu(I)NO as an axial ligand to Type 2 Cu(I).     

Computation of hyperfine tensors for dinuclear Mn(III) Mn(IV) complexes by broken-symmetry approaches: anisotropy transfer induced by local zero-field splitting

Based on broken-symmetry density functional calculations, the (55)Mn hyperfine tensors of a series of exchange-coupled, mixed-valence, dinuclear Mn(III) Mn(IV) complexes have been computed. We go beyond previous quantum chemical work by fully including the effects of local zero-field splitting (ZFS) interactions in the spin projection, following the first-order perturbation formalism of Sage et al. [J. Am. Chem. Soc. 1989, 111, 7239]. This allows the ZFS-induced transfer of hyperfine anisotropy from the Mn(III) site to the Mn(IV) site to be described with full consideration of the orientations of local hyperfine and ZFS tensors. After scaling to correct for systematic deficiencies in the quantum chemically computed local ZFS tensors, good agreement with experimental (55)Mn anisotropies at the Mn(IV) site is obtained. The hyperfine coupling anisotropies on the Mn(III) site depend sensitively on structural distortions for a d(4) ion. The latter are neither fully reproduced by using a DFT-optimized coordination environment nor by using experimental structures. For very small exchange-coupling constants, the perturbation treatment breaks down and a dramatic sensitivity to the scaling of the local ZFS tensors is observed. These results are discussed with respect to ongoing work to elucidate the structure of the oxygen-evolving complex of photosystem II by analysis of the EPR spectra.     

Calculating the electron paramagnetic resonance parameters of exchange coupled transition metal complexes using broken symmetry density functional theory: application to a MnIII/MnIV model compound

The capability of the density functional broken symmetry approach for the calculation of various EPR parameters of exchange coupled metal clusters is demonstrated by studying the experimentally well-investigated [Mn(III)Mn(IV)(mu-O)(2)(mu-OAc)DTNE](2+) complex. Geometry optimizations of the complex in its broken symmetry and high spin states yielded structures with two distinct manganese sites and geometrical parameters in good agreement with the X-ray structure. Exchange coupling constants were calculated from the energy differences between the high spin and broken symmetry states using the Heisenberg spin Hamiltonian. Very good agreement between theory and experiment was achieved with the B3LYP hybrid functional. The g-tensor calculations were performed employing the coupled perturbed Kohn-Sham equations. A strategy for the computation of g-tensor site values is presented and provides single-site g-tensors that are in good agreement with the expectations for Mn(III) and Mn(IV), respectively. Spin projection gave the g-tensor of the coupled manganese complex in very good agreement with the experimental results. Complete (55)Mn hyperfine tensors, including spin-orbit contributions, were calculated and spin-projected. The source of anisotropy in this system could be traced back to the Mn(III) ion in line with the experimental results. The isotropic manganese hyperfine coupling constants were underestimated by factors between 1.4 and 2.5. It is shown that this deficiency is systematic in character and not anchored in the broken symmetry approach. Nuclear quadrupole splitting of the (55)Mn nuclei is shown to be small in this system. In addition, (14)N and (1)H ligand hyperfine data were calculated and compared well with the experimental results. The quality of the extended point-dipole model was demonstrated in application to (1)H anisotropic hyperfine coupling constants.     

Cu[CC6H11O)2PS2]2配合物单晶的电子顺磁共振

本文报道了Cu[(C6H11O)2PS2]2配合物单晶在X波段室温下的电子顺磁共振研究. 电子顺磁共振谱显示出由^6^3Cu和^6^5Cu的磁性核引起的超精细结构以及由配体^3^1P的磁性核引起的配体超精细结构. 用非同轴的g张量和A张量系统的最小二乘拟合技术, 严格地计算了自旋Hamiltonian参数. g张量的主值表征, Cu^2^+处在由四个配体S形成的平行四方形的中心, 具有四角对称性, 但是由于配体中两个P的影响, 在CuS4平面上A张量的主值出现较大的各向异性. g张量和A张量有一个主轴是共轴的, 它们与CuS4平面垂直. 实验上观察到电子自旋与配体中^3^1P的相互作用是各向同性的, 并获得相应的配体超精细耦合常数A^p值.     

High-resolution electron spin resonance spectroscopy of XeF* in solid argon. The hyperfine structure constants as a probe of relativistic effects in the chemical bonding properties of a heavy noble gas atom

Xenon fluoride radicals were generated by solid-state chemical reactions of mobile fluorine atoms with xenon atoms trapped in Ar matrix. Highly resolved electron spin resonance spectra of XeF* were obtained in the temperature range of 5-25 K and the anisotropic hyperfine parameters were determined for magnetic nuclei 19F, 129Xe, and 131Xe using naturally occurring and isotopically enriched xenon. Signs of parallel and perpendicular hyperfine components were established from analysis of temperature changes in the spectra and from numerical solutions of the spin Hamiltonian for two nonequivalent magnetic nuclei. Thus, the complete set of components of hyperfine- and g-factor tensors of XeF* were obtained: 19F (Aiso=435, Adip=1249 MHz) and 129Xe (Aiso=-1340, Adip=-485 MHz); g(parallel)=1.9822 and g(perpendicular)=2.0570. Comparison of the measured hyperfine parameters with those predicted by density-functional theory (DFT) calculations indicates, that relativistic DFT gives true electron spin distribution in the 2Sigma+ ground-state, whereas nonrelativistic theory underestimates dramatically the electron-nuclear contact Fermi interaction (Aiso) on the Xe atom. Analysis of the obtained magnetic-dipole interaction constants (Adip) shows that fluorine 2p and xenon 5p atomic orbitals make a major contribution to the spin density distribution in XeF*. Both relativistic and nonrelativistic calculations give close magnetic-dipole interaction constants, which are in agreement with the measured values. The other relativistic feature is considerable anisotropy of g-tensor, which results from spin-orbit interaction. The orbital contribution appears due to mixing of the ionic 2Pi states with the 2Sigma+ ground state, and the spin-orbit interaction plays a significant role in the chemical bonding of XeF*.     

Electronic structure of binuclear mixed valence copper azacryptates derived from integrated advanced EPR and DFT calculations

Binuclear, mixed valence copper complexes with a [Cu(+1)(.5), Cu(+1)(.5)] redox state and S = (1)/(2) can be stabilized with rigid azacryptand ligands. In this system the unpaired electron is delocalized equally over the two copper ions, and it is one of the very few synthetic models for the electron mediating Cu(A) site of nitrous oxide reductase and cytochrome c oxidase. The spatial and electronic structures of the copper complex in frozen solution were obtained from the magnetic interactions, namely the g-tensor and the (63,65)Cu, (14)N, (2)H, and (1)H hyperfine couplings, in combination with density functional theory (DFT) calculations. The magnetic interactions were determined from continuous wave (CW) electron paramagnetic resonance (EPR), pulsed electron nuclear double resonance (ENDOR), two-dimensional TRIPLE, and hyperfine sublevel correlation spectroscopy (HYSCORE) carried out at W-band or/and X-band frequencies. The DFT calculated g and Cu hyperfine values were in good agreement with the experimental values showing that the structure in solution is indeed close to that of the optimized structure. Then, the DFT calculated hyperfine parameters were used as guidelines and starting points in the simulations of the various experimental ENDOR spectra. A satisfactory agreement with the experimental results was obtained for the (14)N hyperfine and quadrupole interactions. For (1)H the DFT calculations gave good predictions for the hyperfine tensor orientations and signs, and they were also successful in reproducing trends in the magnitude of the various proton hyperfine couplings. These, in turn, were very useful for ENDOR signals assignments and served as constraints on the simulation parameters.     

Automatic fitting to 'powder' EPR spectra of coupled paramagnetic species employing Feynman's theorem

A previous automatic fitting procedure of EPR spectra has been extended with the purpose to characterise coupled paramagnetic complexes in powders and frozen solutions. The theoretical EPR spectra were obtained by matrix diagonalization of a general spin Hamiltonian. A least-squares fitting procedure using analytical derivatives of the calculated spectrum with respect to the spectroscopic, fine structure, nuclear quadrupole, electron-electron, and hyperfine coupling tensors was used to refine those parameters. The powder spectra of matrix isolated *CF3 and RCF2CF2* radicals, previously measured at low temperature, were reanalysed with this method. A theoretically modeled complex consisting of a Cu2+ ion, featuring an axially symmetric g-tensor and 63Cu hyperfine structure anisotropy, and a free radical located at different orientations, with respect to the symmetry axis of the Cu2+ ion, was examined in order to investigate the possibility to recover the magnetic parameters of the separate units and the magnetic couplings between them.