The spectra were recorded on a prototypical Elexysys® E780 from Bruker Biospin. The 263 GHz spectrometer works with a quasi optical front end. The front end produces a Gaussian beam that is focused to a corrugated waveguide. The typical output power of the bridge was 15 mW. The corrugated waveguide is coupled to a single mode (TE011) cylindrical cavity (E9501610) with a typical quality factor (Q) of 500-1000. The electron spin echo (π/2-τ-π-τ-echo, ESE) was recorded with a typical microwave field strength B1 of 10-17 MHz. The ESE spectra were recorded by 70 K, if not stated otherwise. The individual B1 is measured via the pulse length necessary for inversion of the magnetization (πtp) by a nutation recorded by an inversion recovery experiment (πtp-T-π/2-τ-π-τ-echo) scanning
over the pulse length of πtp. A standard coal sample is used. The microwave field strength can
Freeze quench samples in 0.33 mm EPR tubes were inserted under liquid nitrogen into the resonator surrounded by liquid nitrogen and then transferred into the precooled (80 K) EPR cryostat (Oxford Instruments).
To assess the accuracy of the g values several error contributions have to be considered. As reported for other high frequency spectrometer258 the frequency change over a measurement is not a significant source of error. The spectrometer has a sweep coil with 250 mT range and the main magnet operating until 12 T. If the main magnetic field is changed a systematic change of all g values has to be considered. Therefore, a reliable calibration of the field is necessary. Field calibration was originally performed based on a multiline Mn(II) standard sample, by Bruker. Normally, the Mn(II)(0.02% in MgO) has been used with a g value of 2.001015(5) and the HF coupling A = -243.9 (1) MHz.259, 260 The Mn(II) in marble used by Bruker and Jeol has different values with g =2.0011 and A= -241.6 MHz.261, 262 With this standard sample a single field point can be calibrated and the linearity of the sweep coil sweep is evaluated by the 6 lines of the 55Mn hyperfine interaction within the Kramers doublet. The non-linearity originates mainly from the self-inductance of the sweep coil, and the mutual inductance of the sweep and main magnet coil.258 To compensate this non-linear behavior of the ratio between gauss to amp, Bruker implemented a linearization protocol for magnetic field sweeps in CW-EPR spectra. In this protocol the typically small measurement range for organic radicals (20-50 mT) is always measured by a full sweep range of the sweep coil. Here the curve difference to a linear behavior can be approximated by a second order fit (cf. Figure 3-2). Hence, the sweep coil has to be driven through the full (250 mT) sweep range. This extends the measurement time by a factor of 5 to13 depending on the sweep range. To be able to omit this large drawback, the impact on the spectral accuracy of this linearization option was evaluated. For this reason, the CW-EPR spectra of the manganese standard sample in increasing field direction were measured with and without the linearization procedure. After aligning the first line (Figure 3-1 inset left) the shift was measured on each line position. Thus, the scan over the six lines performed with the linearization procedure is about 4.4 G narrower than one without linearization. This gives an estimate of the systematic error introduced by removing
263 GHz Spectroscopy and Calibration
the linearization procedure and reducing the measurement time. The additional error for a typical field sweep of 30 mT is 0.36 mT based on a fit of the data (Figure 3-2). Compared to the typical field of 9350 mT, this error is 3.9∙10-5 mT. Due to the g factor of approximately two (for organic radicals in this thesis), a systematic error of 8∙10-5 is obtained. The overall systematic shift in the ampere to gauss ratio has to be re-adjusted from time to time, to align with the standard values with the setting. However, the standard sample used here could be only observed by CW-EPR with a sharp line width of ≈0.12 mT. Therefore, another sample was necessary in order to test the pulsed set up.
Figure 3-1: Comparison of CW 263-GHz EPR spectra of Mn2+ (in CaO, 0.02%) at room temperature with and without linearization. Exp. details: Field sweep range = 100 mT;
modulation amplitude = 0.5 G; conversion time=100 ms; single scan.
Figure 3-2: Linearization improvement compared in CW 263 GHz EPR measurements. The difference in magnetic field between the linearized and the non-linearized field sweep as ΔB0 is plotted against the width of the Mn2+ resonance lines. The points can be fitted with a second order polynomial(red line) with: y=0.148(1)∙x - 9.2(4) ∙10-6∙x2 and R2=0.99994.
To test the field accuracy in the pulsed mode the β2-Y122• E. coli RNR sample was used, which is well characterized at high-field EPR. An advantage of taking the Y122• as a standard is that it can be used as an internal standard for the RNR samples studied. This internal standard is detectable at 10 K and is hidden at elevated temperatures (70 K).55, 148, 152 The derivative has to be formed to assign the principle axis values of gx, gy and gz in the g tensor broadened line. The spectrum of Y122• has been recorded at 10 K. The spectrum was then compared to high-field powder157 and crystal data63 of Y122•. Högbom et al. have used as a calibration standard a narrow single line Li:F g-standard (Li in LiF, g = 2.002293±0.000002263) measured at two different frequencies.63 Gerfen et al. used multiline Mn(II)(0.02% in MgO)264 with a g value of 2.001015(5) and the HF coupling A = -244.1 (1) MHz.157, 259, 260 The Y122• spectrum measured at our instrument was compared to a simulation based on these two literature values, as shown in Figure 3-3. The simulation parameters reported by Gerfen (gray) et al. show an agreement with the spectrum in terms of g values and deviate from those reported by Högbom et al. (green) at gz and gy by 2∙10-5 and 6∙10-5, respectively. In both experiments an error of 4∙10-5 or 5∙10-5 was estimated.63, 84
263 GHz Spectroscopy and Calibration
Figure 3-3: Pulsed-EPR spectrum of the β2-Y122• as calibration standard. The experimental trace is shown in blue and the simulation gray and green with values from 157 and 63, respectively.
Exp. details: ESE, 262.0109 GHz, T=10 K, π(π/2)= 52(90) ns, τ=319 ns, shots per point (SPP)=50, shot repetition time (SRT)=15 ms, scans = 43. The derivative was built by a Savitzky-Golay filter (second order, 3 points).
Another calibration with a N@C60 sample265 (from A. Schneggs lab at the Helmholtz Zentrum, Berlin) was performed recently by I. Tkach in our group. He found a standard deviation within 8 resonance frequencies averaged over three line measurements to be in g 3.3∙10-6. By reducing the sweep range from 60 mT by a factor of 10 a systematic shift of -1.5∙10-5 could be observed, based on 3 observing frequencies and 12 measured resonances. The change of other parameters by a factor of ten, like a tenfold increase in sweep time gave no significant shifts. All of these errors are far below the errors reported within this thesis and are therefore neglected in the future discussion. Based on the measurements, the systematic error with or without linearization procedure in CW and pulsed EPR spectroscopy can be estimated. The error estimated by the differences observed here is below 5∙10-5 in g scale. For experiments where field linearization has not been used a
systematic error of 9∙10-5 is estimated. However, due to low signal to noise ratios (S/N) and broad line widths, the g value uncertainty can vary in an individual spectrum.
3.6 Density Functional Theory Calculations 3.6.1 Set-up of the Models
DFT calculations have been performed with the ORCA 3.0.0 program package214. DFT calculations were originally performed by Christoph Riplinger (ORCA 2.9.0) from the Neese group and had been performed as previously reported.110 The geometry-optimized large models are based on the crystal structure (wt-α, PDB ID 4R1R) and had shown by energy-optimized relaxed surface scans, along the reaction coordinate, energy barriers in agreement or lower than previously reported values with smaller models by DFT theorists.106, 107, 266 To compare these models to experimental findings, Simone Kossmann from the Neese group incorporated the amino group at Y731• and re-optimized the geometry.
The EPR parameters were calculated by Simone Kossmann. The adaptation into magnetic resonance convention and the interpretation of the output was done by me. Small model calculations have been performed to test different environment dependencies of the structurally ill-defined region.
3.6.2 Geometry Optimizations
3.6.2.1 Large Models 1, 2 and 3
Initially, the coordinates of the large models 7 and 8 used in ref110 augmented by the amino group in the 3 position of Y731 and a water molecule between Y731 and Y730 for Model 3.
These coordinates were first geometry optimized without further restraints. During the optimization the distance between C439 and Y730 increased constantly. It was supposed this results from the missing contact to the β subunit in the model, thus the coordinates were restrained for all Cα and for all Cβ. For Y730, NH2Y731 and C439 only the Cα were restrained.
Additionally the Cartesian coordinates of the hydrogen atoms in the truncated GPD model replacing the bonds between C4 and C5 of the ribose as well as the bond between C1 of the ribose and the base were kept fixed.
The model structures were geometry optimized using a generalized gradient density
Density Functional Theory Calculations
of triple-ζ quality207, 269. Grimme’s dispersion correction200, 270 has been now added on top of the SCF calculation. The Resolution of the Identity (RI) approximation with the corresponding auxiliary basis sets (def2-TZVP(P)/JK150) has been employed throughout.
3.6.2.2 Small Models
In the small models the geometry optimization was performed on the B3LYP268, 271, 272 hybrid density functional in combination with the TZVPP basis set and def2-TZVPP/JK auxiliary basis set. In the models adapted from the large models only the dihedral angle of the peptide bond of Y730 and Y731 was fixed and the Cartesian restraints for all surrounding Cα’s were kept. In order to compensate the electrostatics from the environment here a solvation model (COSMO273) with polarity of ethanol (ε=24) was used. Otherwise Grimme’s dispersion correction199-201 and RIJCOSX274 approximations has been employed. The energy has been converged to 10-9 Eh, if not stated otherwise.
3.6.3 EPR Calculations
The EPR calculations were carried out with the B3LYP268, 271, 272 hybrid density functional in combination with the RIJCOSX274 approximation. In the small models COSMO was retained for the single point calculations. Here Barone’s EPR-II (IGLO-II for sulfur) basis set of double-ζ quality has been used in combination with the def2-TZVPP/JK auxiliary basis set for all atoms.219, 220, 275 The g values were calculated210 using the tyrosine (analog) C4
as gauge origin. In single amino acid models the def2-TZVPP basis set was held consistent with the geometry optimization step.207 The dihedral scans were performed with a geometry optimization for each restrained dihedral. The energy has been converged to 10-9 Eh.