Edgar J. J. Groenen

Distance measurements in the borderline region of applicability of CW EPR and DEER: A model study on a homologous series of spin-labelled peptides

Inter-spin distances between 1 nm and 4.5 nm are measured by continuous wave (CW) and pulsed electron paramagnetic resonance (EPR) methods for a series of nitroxide-spin-labelled peptides. The upper distance limit for measuring dipolar coupling by the broadening of the CW spectrum and the lower distance limit for the present optimally-adjusted double electron electron resonance (DEER) set-up are determined and found to be both around 1.6-1.9 nm. The methods for determining distances and corresponding distributions from CW spectral line broadening are reviewed and further developed. Also, the work shows that a correction factor is required for the analysis of inter-spin distances below approximately 2 nm for DEER measurements and this is calculated using the density matrix formalism.

On the fluorescence of crystalline C60 at 1.2 K

Abstract Well-resolved fluorescence spectra are reported for single crystals of C60 at 1.2 K. The fluorescence is found to be composed of two contributions. Fluorescence-microwave double-resonance experiments indicate that the dominant component originates from deep X-traps that consists of pairs of C60 molecules. The minor component most likely derives from shallow monomolecular C60 X-traps.

Triplet excitation of C60 and the structure of the crystal at 1.2 K

Abstract Electron-spin-echo experiments at 95 GHz and 1.2 K reveal that the triplet excitation of a single crystal of C60 is delocalized over either a pair or a chain of C60 molecules along 〈110〉 directions. Two distinct triplet species have been observed in a one-to-one ratio which verifies the proposed 2 a0 face-centred cubic superstructure of the crystal at low temperature. The C60 molecules on the two sublattices differ by a rotation over 60° about 〈111〉 directions.

Phosphorescence of C60 at 1.2 K

Abstract Phosphorescence has been observed at 1.2 K both for C 60 dissolved in a decaline/cyclohexane glass and for single crystals of C 60 . The phosphorescence spectrum of C 60 in the glass reveals a weak origin at 798.1 ± 0.5 nm, while most of the intensity shows up in vibronic transitions. The phosphorescence of crystalline C 60 consists of two parts. One of these, with the origin of its phosphorescence spectrum at 826 nm, derives probably from a shallow C 60 X-trap although an excitonic origin cannot be excluded. The other, with the origin of its phosphorescence spectrum at 862 nm, originates from triplet states previously recognized by magnetic resonance experiments. These concern excitations delocalized over pairs of C 60 molecules, so-called mini-excitons that are deep X-traps in the C 60 crystal.

Single Molecule Electron Paramagnetic Resonance Spectroscopy: Hyperfine Splitting Owing to a Single Nucleus

Individual pentacene-d14 molecules doped into a p-terphenyl-d14 host crystal have been studied by optically detected electron paramagnetic resonance spectroscopy. The magnetic resonance transitions between the triplet sublevels of the pentacene molecule and the splitting of the resonance lines for a molecule that contains a carbon-13 nucleus have been observed in an external magnetic field. This splitting is caused by the hyperfine interaction of the triplet electron spin with the single carbon-13 nuclear spin.

A Multifrequency High-Field Electron Paramagnetic Resonance Study of CoIIS4 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.

Spectroscopic Characterization of the Electronic Changes in the Active Site of Streptomyces antibioticus Tyrosinase upon Binding of Transition State Analogue Inhibitors

The dinuclear copper enzyme tyrosinase (Ty) from genetically engineered Streptomyces antibioticus has been investigated in its paramagnetic half-met form [Cu(I)-Cu(II)]. The cw EPR, pulsed EPR, and hyperfinesublevel correlation spectroscopy (HYSCORE) experiments on the half-met-Ty and on its complexes with three different types of competitive inhibitor are reported. The first type includes p-nitrophenol, a very poor substrate for the monooxygenase activity of Ty. The second type comprises hydroxyquinones, such as kojic acid and l-mimosine, and the third type of inhibitor is represented by toluic acid. The electronic and structural differences of the half-met-Ty form induced at the cupric site by the different inhibitors have been determined. Probes of structural effects are the hyperfine coupling constants of the non coordinating Nδ histidyl nitrogens. By using the available crystal structures of hemocyanin as a template in combination with the spectroscopic results, a structural model for the active site of half-met-Ty is obtained and a model for the binding modes of both mono- and diphenols could be proposed.

EPR study of the dinuclear active copper site of tyrosinase from Streptomyces antibioticus

The [Cu(I)–Cu(II)] half‐met form of the dinuclear copper site of tyrosinase has been probed by continuous wave electron paramagnetic resonance (EPR) and hyperfine sublevel correlation (HYSCORE) spectroscopy in the presence and absence of inhibitors. In all cases the EPR spectrum is indicative of a dx 2−y 2 ground state for the unpaired electron. From the cross‐peaks observed in the HYSCORE spectra, proton hyperfine coupling constants were obtained that are compatible with a hydroxide ion in an equatorial coordination position of the paramagnetic copper. After changing the water solvent to D2O or after addition of the inhibitors p‐nitrophenol or L‐mimosine, the proton signals disappear. The relevance of these findings for understanding the catalytic cycle is discussed.

An ENDOR and ESEEM study of the blue copper protein azurin

Abstract We report electron-nuclear double-resonance experiments on a single crystal of azurin at 95 GHz and electron-spin-echo envelope-modulation experiments on frozen solutions of azurin and of the H117G mutant at 9 GHz. The hyperfine and quadrupole tensors of the two remote nitrogens of the histidine ligands of copper are assigned and discussed. A third nucleus is found to contribute to the echo-modulation spectrum and this probably concerns an amide nitrogen of the peptide backbone.

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 Cus in the trinuclear center, as noted before, but also to the enzyme mechanism.