17 O Hyperfine Spectroscopy Reveals Hydration Structure of Nitroxide Radicals in Aqueous Solutions.

The hydration structure of nitroxide radicals in aqueous solutions is elucidated by advanced 17 O hyperfine (hf) spectroscopy with support of quantum chemical calculations and MD simulations. A piperidine and a pyrrolidine-based nitroxide radical are compared and show clear differences in the preferred directionality of H-bond formation. We demonstrate that these scenarios are best represented in 17 O hf spectra, where in-plane coordination over σ ${\sigma }$ -type H-bonding leads to little spin density transfer on the water oxygen and small hf couplings, whereas π ${{\rm \pi }}$ -type perpendicular coordination generates much larger hf couplings. Quantitative analysis of the spectra based on MD simulations and DFT predicted hf parameters is consistent with a distribution of close solvating water molecules, in which directionality is influenced by subtle steric effects of the ring and the methyl group substituents.

19F Electron-Nuclear Double Resonance Reveals Interaction between Redox-Active Tyrosines across the α/ß Interface of E. coli Ribonucleotide Reductase.

Ribonucleotide reductases (RNRs) catalyze the reduction of ribonucleotides to deoxyribonucleotides, thereby playing a key role in DNA replication and repair. Escherichia coli class Ia RNR is an α2ß2 enzyme complex that uses a reversible multistep radical transfer (RT) over 32 Å across its two subunits, α and ß, to initiate, using its metallo-cofactor in ß2, nucleotide reduction in α2. Each step is proposed to involve a distinct proton-coupled electron-transfer (PCET) process. An unresolved step is the RT involving Y356(ß) and Y731(α) across the α/ß interface. Using 2,3,5-F3Y122-ß2 with 3,5-F2Y731-α2, GDP (substrate) and TTP (allosteric effector), a Y356• intermediate was trapped and its identity was verified by 263 GHz electron paramagnetic resonance (EPR) and 34 GHz pulse electron-electron double resonance spectroscopies. 94 GHz 19F electron-nuclear double resonance spectroscopy allowed measuring the interspin distances between Y356• and the 19F nuclei of 3,5-F2Y731 in this RNR mutant. Similar experiments with the double mutant E52Q/F3Y122-ß2 were carried out for comparison to the recently published cryo-EM structure of a holo RNR complex. For both mutant combinations, the distance measurements reveal two conformations of 3,5-F2Y731. Remarkably, one conformation is consistent with 3,5-F2Y731 within the H-bond distance to Y356•, whereas the second one is consistent with the conformation observed in the cryo-EM structure. The observations unexpectedly suggest the possibility of a colinear PCET, in which electron and proton are transferred from the same donor to the same acceptor between Y356 and Y731. The results highlight the important role of state-of-the-art EPR spectroscopy to decipher this mechanism.

Trapping X-ray Radiation Damage from Homolytic Se-C Bond Cleavage in BnSeSeBn Crystals (Bn=benzyl, CH2 C6 H5 ).

Irradiation of dibenzyl diselenide BnSeSeBn with X-ray or UV-light cleaves the Se-C and the Se-Se bonds, inducing stable and metastable radical states. They are inevitably important to all natural and life sciences. Structural changes due to X-ray-induced Se-C bond-cleavage could be pin-pointed in various high-resolution X-ray diffraction experiments for the first time. Extended DFT methods were applied to characterize the solid-state structure and support the refinement of the observed residuals as contributions from the BnSeSe⋅ radical species. The X-ray or UV-irradiated crystalline samples of BnSeSeBn were characterized by solid-state EPR. This paper provides insight that in the course of X-ray structure analysis of selenium compounds not only organo-selenide radicals like RSe⋅ may occur, but also organo diselenide BnSeSe⋅ radicals and organic radicals R⋅ are generated, particularly important to know in structural biology.

Resolution of chemical shift anisotropy in 19F ENDOR spectroscopy at 263 GHz/9.4 T.

Pulsed 19F ENDOR spectroscopy provides a selective method for measuring angstrom to nanometer distances in structural biology. Here, the performance of 19F ENDOR at fields of 3.4 T and 9.4 T is compared using model compounds containing one to three 19F atoms. CF3 groups are included in two compounds, for which the possible occurrence of uniaxial rotation might affect the distance distribution. At 9.4 T, pronounced asymmetric features are observed in many of the presented 19F ENDOR spectra. Data analysis by spectral simulations shows that these features arise from the chemical shift anisotropy (CSA) of the 19F nuclei. This asymmetry is also observed at 3.4 T, albeit to a much smaller extent, confirming the physical origin of the effect. The CSA parameters are well consistent with DFT predicted values and can be extracted from simulation of the experimental data in favourable cases, thereby providing additional information about the geometrical and electronic structure of the spin system. The feasibility of resolving the CSA at 9.4 T provides important information for the interpretation of line broadening in ENDOR spectra also at lower fields, which is relevant for developing methods to extract distance distributions from 19F ENDOR spectra.

Detection of Water Molecules on the Radical Transfer Pathway of Ribonucleotide Reductase by 17O Electron-Nuclear Double Resonance Spectroscopy.

The role of water in biological proton-coupled electron transfer (PCET) is emerging as a key for understanding mechanistic details at atomic resolution. Here we demonstrate 17O high-frequency electron-nuclear double resonance (ENDOR) in conjunction with H217O-labeled protein buffer to establish the presence of ordered water molecules at three radical intermediates in an active enzyme complex, the α2ß2 E. coli ribonucleotide reductase. Our data give unambiguous evidence that all three, individually trapped, intermediates are hyperfine coupled to one water molecule with Tyr-O···17O distances in the range 2.8-3.1 Å. The availability of this structural information will allow for quantitative models of PCET in this prototype enzyme. The results also provide a spectroscopic signature for water H-bonded to a tyrosyl radical.

1H high field electron-nuclear double resonance spectroscopy at 263 GHz/9.4 T.

We present and discuss the performance of 1H electron-nuclear double resonance (ENDOR) at 263 GHz/9.4 T by employing a prototype, commercial quasi optical spectrometer. Basic instrumental features of the setup are described alongside a comprehensive characterization of the new ENDOR probe head design. The performance of three different ENDOR pulse sequences (Davies, Mims and CP-ENDOR) is evaluated using the 1H BDPA radical. A key feature of 263 GHz spectroscopy - the increase in orientation selectivity in comparison with 94 GHz experiments - is discussed in detail. For this purpose, the resolution of 1H ENDOR spectra at 263 GHz is verified using a representative protein sample containing approximately 15 picomoles of a tyrosyl radical. Davies ENDOR spectra recorded at 5 K reveal previously obscured spectral features, which are interpreted by spectral simulations aided by DFT calculations. Our analysis shows that seven internal proton couplings are detectable for this specific radical if sufficient orientation selectivity is achieved. The results prove the fidelity of 263 GHz experiments in reporting orientation-selected 1H ENDOR spectra and demonstrate that new significant information can be uncovered in complex molecular systems, owing to the enhanced resolution combined with high absolute sensitivity and no compromise in acquisition time.

The Guinness molecules for the carbohydrate formula.

A systematic review and analysis of the most stable spatial arrangements of n carbon, n oxygen, and 2n hydrogen atoms including vibrational zero-point energy up to n = 5 shows that small-molecule aggregates win, typically followed by thermally unstable molecules, before kinetically stable molecules and finally carbohydrates are found. Near n ≈ 60 a crossover to carbon allotropes and ice as the global minimum structure is expected and the asymptotic limit is most likely graphite and ice. Implications for astrochemical and fermentation processes are discussed. Density functionals like B3LYPD3 are found to describe these energy sequences quite poorly, mostly due to an overestimated stability of carbon in high oxidation states.