Exploring tunneling ESEEM beyond methyl groups in nitroxides at low temperatures.

Tunneling of methyl rotors coupled to an electron spin causes magnetic field independent electron spin echo envelope modulation (ESEEM) at low temperatures. For nitroxides containing alkyl substituents, we observe this effect as a contribution at the beginning of the Hahn echo decay signal occurring on a faster time scale than the matrix-induced decoherence. The tunneling ESEEM contribution includes information on the local environment of the methyl rotors, which manifests as a distribution of rotation barriers P(V3) when measuring the paramagnetic species in a glassy matrix. Here, we investigate the differences in tunneling behaviour of geminal methyl and ethyl group rotors in nitroxides while exploring different levels of theory in our previously introduced methyl quantum rotor (MQR) model. Moreover, we extend the MQR model to analyze the tunneling ESEEM originating from two different rotor types coupled to the same electron spin. We find that ethyl groups in nitroxides give rise to stronger tunneling ESEEM contributions than methyl groups because the difference between hyperfine couplings of their methyl protons better matches the tunneling frequency. The methyl rotors of both ethyl and propyl groups exhibit distributions at lower rotation barriers compared to geminal methyl groups. This is in good agreement with density functional theory (DFT) calculations of their rotation barriers and showcases that conformational flexibility impacts the hindrance of rotation. Using Monte-Carlo based fitting in combination with an identifiability analysis of the MQR model parameter space, we extract rotation barrier distributions for the individual rotor types in mixed-rotor nitroxides as well as identify which rotors dominate the observed tunneling contribution in the Hahn echo decay signal.

Identification of a poly-cyclopropylglycine-containing peptide via bioinformatic mapping of radical S-adenosylmethionine enzymes.

Peptide-derived natural products are a large class of bioactive molecules that often contain chemically challenging modifications. In the biosynthesis of ribosomally synthesized and posttranslationally modified peptides (RiPPs), radical-SAM (rSAM) enzymes have been shown to catalyze the formation of ether, thioether, and carbon-carbon bonds on the precursor peptide. The installation of these bonds typically establishes the skeleton of the mature RiPP. To facilitate the search for unexplored rSAM-dependent RiPPs for the community, we employed a bioinformatic strategy to screen a subfamily of peptide-modifying rSAM enzymes which are known to bind up to three [4Fe-4S] clusters. A sequence similarity network was used to partition related families of rSAM enzymes into >250 clusters. Using representative sequences, genome neighborhood diagrams were generated using the Genome Neighborhood Tool. Manual inspection of bacterial genomes yielded numerous putative rSAM-dependent RiPP pathways with unique features. From this analysis, we identified and experimentally characterized the rSAM enzyme, TvgB, from the tvg gene cluster from Halomonas anticariensis. In the tvg gene cluster, the precursor peptide, TvgA, is comprised of a repeating TVGG motif. Structural characterization of the TvgB product revealed the repeated formation of cyclopropylglycine, where a new bond is formed between the γ-carbons on the precursor valine. This novel RiPP modification broadens the functional potential of rSAM enzymes and validates the proposed bioinformatic approach as a practical broad search tool for the discovery of new RiPP topologies.

Cucurbit[7]uril Enhances Distance Measurements of Spin-Labeled Proteins.

We report complex formation between the chloroacetamide 2,6-diazaadamantane nitroxide radical (ClA-DZD) and cucurbit[7]uril (CB-7), for which the association constant in water, Ka = 1.9 × 106 M-1, is at least 1 order of magnitude higher than the previously studied organic radicals. The radical is highly immobilized by CB-7, as indicated by the increase in the rotational correlation time, τrot, by a factor of 36, relative to that in the buffer solution. The X-ray structure of ClA-DZD@CB-7 shows the encapsulated DZD guest inside the undistorted CB-7 host, with the pendant group protruding outside. Upon addition of CB-7 to T4 Lysozyme (T4L) doubly spin-labeled with the iodoacetamide derivative of DZD, we observe the increase in τrot and electron spin coherence time, Tm, along with the narrowing of interspin distance distributions. Sensitivity of the DEER measurements at 83 K increases by a factor 4-9, compared to the common spin label such as MTSL, which is not affected by CB-7. Interspin distances of 3 nm could be reliably measured in water/glycerol up to temperatures near the glass transition/melting temperature of the matrix at 200 K, thus bringing us closer to the goal of supramolecular recognition-enabled long-distance DEER measurements at near physiological temperatures. The X-ray structure of DZD-T4L 65 at 1.12 Å resolution allows for unambiguous modeling of the DZD label (0.88 occupancy), indicating an undisturbed structure and conformation of the protein.

Synthesis and characterization of a biocompatible 13C1 isotopologue of trityl radical OX071 for in vivo EPR viscometry.

We describe the synthesis, characterization, and application of an isotopologue of the trityl radical OX071, labeled with 13C at the central carbon (13C1). This spin probe features large anisotropy of the hyperfine coupling with the 13C1 (I = 1/2), leading to an EPR spectrum highly sensitive to molecular tumbling. The high biocompatibility and lack of interaction with blood albumin allow for systemic delivery and in vivo measurement of tissue microviscosity by EPR.

Bis-Spiro-Oxetane and Bis-Spiro-Tetrahydrofuran Pyrroline Nitroxide Radicals: Synthesis and Electron Spin Relaxation Studies.

Synthesis of bis-spiro-oxetane and bis-spiro-tetrahydrofuran pyrroline nitroxide radicals relies on the Mitsunobu reaction-mediated double cyclizations of N-Boc protected pyrroline tetraols. Structures of the nitroxide radicals are supported by X-ray crystallography. In a trehalose/sucrose matrix at room temperature, the bis-spiro-oxetane nitroxide radical possesses electron spin coherence time, Tm ≈ 0.7 µs. The observed enhanced Tm is most likely associated with strong hydrogen bonding of oxetane moieties to the trehalose/sucrose matrix.

Spectroscopic and Electrochemical Characterization of the Mycofactocin Biosynthetic Protein, MftC, Provides Insight into Its Redox Flipping Mechanism.

Mycofactocin is a putative redox cofactor and is classified as a ribosomally synthesized and post-translationally modified peptide (RiPP). Some RiPP natural products, including mycofactocin, rely on a radical S-adenosylmethionine (RS, SAM) protein to modify the precursor peptide. Mycofactocin maturase, MftC, is a unique RS protein that catalyzes the oxidative decarboxylation and C-C bond formation on the precursor peptide MftA. However, the number, chemical nature, and catalytic roles for the MftC [Fe-S] clusters remain unknown. Here, we report that MftC binds a RS [4Fe-4S] cluster and two auxiliary [4Fe-4S] clusters that are required for MftA modification. Furthermore, electron paramagnetic resonance spectra of MftC suggest that SAM and MftA affect the environments of the RS and Aux I cluster, whereas the Aux II cluster is unaffected by the substrates. Lastly, reduction potential assignments of individual [4Fe-4S] clusters by protein film voltammetry show that their potentials are within 100 mV of each other.

Structural, Biochemical, and Bioinformatic Basis for Identifying Radical SAM Cyclopropyl Synthases.

The importance of radical S-adenosyl-l-methionine (RS) enzymes in the maturation of ribosomally synthesized and post-translationally modified peptides (RiPPs) continues to expand, specifically for the RS-SPASM subfamily. We recently discovered an RS-SPASM enzyme that installs a carbon-carbon bond between the geminal methyls of valine residues, resulting in the formation of cyclopropylglycine (CPG). Here, we sought to define the family of cyclopropyl (CP) synthases because of the importance of cyclopropane scaffolds in pharmaceutical development. Using RadicalSAM.org, we bioinformatically expanded the family of CP synthases and assigned unique peptide sequences to each subclade. We identified a unique RiPP biosynthetic pathway that encodes a precursor peptide, TigB, with a repeating TIGSVS motif. Using LCMS and NMR techniques, we show that the RS enzyme associated with the pathway, TigE, catalyzes the formation of a methyl-CPG from the conserved isoleucine residing in the repeating motif of TigB. Furthermore, we obtained a crystal structure of TigE, which reveals an unusual tyrosyl ligation to the auxiliary I [4Fe-4S] cluster, provided by a glycine-tyrosine-tryptophan motif unique to all CP synthases. Further, we show that this unique tyrosyl ligation is absolutely required for TigE activity. Together, our results provide insight into how CP synthases perform this unique reaction.

Cucurbit[7]uril Enhances Distance Measurements of Spin-Labeled Proteins.

We report complex formation between the chloroacetamide 2,6-diazaadamantane nitroxide radical (ClA-DZD) and cucurbit[7]uril (CB-7), for which the association constant in water, Ka = 1.9 × 106 M-1, is at least one order of magnitude higher than the previously studied organic radicals. The radical is highly immobilized by CB-7, as indicated by the increase of the rotational correlation time, τrot, by a factor of 36, relative to that in the buffer solution. The X-ray structure of ClA-DZD@CB-7 shows the encapsulated DZD guest inside the undistorted CB-7 host, with the pendant group protruding outside. Upon addition of CB-7 to T4 Lysozyme (T4L) doubly spin-labeled with the iodoacetamide derivative of DZD, we observe the increase in τrot and electron spin coherence time, Tm, along with the narrowing of inter-spin distance distributions. Sensitivity of the DEER measurements at 83 K increases by a factor 4 - 9, compared to the common spin label such as MTSL, which is not affected by CB-7. Inter-spin distances of 3-nm could be reliably measured in water/glycerol up to temperatures near the glass transition/melting temperature of the matrix at 200 K, thus bringing us closer to the goal of supramolecular recognition-enabled long-distance DEER measurements at near physiological temperatures. The X-ray structure of DZD-T4L 65 at 1.12 Å resolution allows for unambiguous modeling of the DZD label (0.88 occupancy), indicating undisturbed structure and conformation of the protein.

Electron paramagnetic resonance characterization and electron spin relaxation of manganate ion in glassy alkaline LiCl solution and doped into Cs2SO4.

Manganate ion, MnO42-, has important roles in catalysis and potential roles in water treatment. EPR spectra of MnO42- in a glassy alkaline solution of concentrated LiCl at X-band and Q-band at 80 K exhibit g1 = 1.9776 ± 0.001, g2 = 1.9677 ± 0.001, g3 = 1.9560 ± 0.001 and A1 = 182 ± 9, A2 = 275 ± 15, and A3 = 400 ± 15 MHz. In Cs2SO4 the spectra were simulated with 1.908 ± 0.001, g2 = 1.909 ± 0.001, g3 = 1.937 ± 0.001 and A1 = 90 ± 20, A2 = 100 ± 20, and A3 = 400 ± 15 MHz. Simulations required large distributions in A values which suggests that hyperfine splittings are sensitive to differences in geometry. Continuous wave spectra are observable at 80 K in glassy alkaline LiCl, but only up to about 20 K in Cs2SO4. In glassy alkaline LiCl electron spin relaxation was measured at X-band using spin echo and inversion recovery from 4.2 to 60 K. Tm is 4.6 µs at 4.2 K and decreases at higher temperatures as it becomes driven by T1. T1 decreases from ca. 34 ms at 4.2 K to ca. 240 ns at 60 K. Tm and T1 in Cs2SO4 are too short to measure by electron spin echo. The distorted tetrahedral geometry of MnO42- results in faster relaxation than for other 3d1 spin systems that have square pyramidal (C4v) or distorted octahedral geometries.

Ligand control of low-frequency electron paramagnetic resonance linewidth in Cr(III) complexes.

Understanding how the ligand shell controls low-frequency electron paramagnetic resonance (EPR) spectroscopic properties of metal ions is essential if they are to be used in EPR-based bioimaging schemes. In this work, we probe how specific variations in the ligand structure impact L-band (ca. 1.3 GHz) EPR spectroscopic linewidths in the trichloride salts of five Cr(iii) complexes: [Cr(RR-dphen)3]3+ (RR-dphen = (1R,2R)-(+)-diphenylethylenediamine, 1), [Cr(en)3]3+ (en = ethylenediamine, 2), [Cr(me-en)3]3+ (me-en = 1,2-diaminopropane, 3), [Cr(tn)3]3+ (tn = 1,3-diaminopropane, 4) [Cr(trans-chxn)3]3+ (trans-chxn = trans-(±)-1,2-diaminocyclohexane, 5). Spectral broadening varies in a nonintuitive manner across the series, showing the sharpest peaks for 1 and broadest for 5. Molecular dynamics simulations provide evidence that the broadening is correlated to rigidity in the inner coordination sphere and reflected in ligand-dependent distribution of Cr-N bond distances that can be found in frozen solution.

Supramolecular Approach to Electron Paramagnetic Resonance Distance Measurement of Spin-Labeled Proteins.

We demonstrate a host-guest molecular recognition approach to advance double electron-electron resonance (DEER) distance measurements of spin-labeled proteins. We synthesized an iodoacetamide derivative of 2,6-diazaadamantane nitroxide (DZD) spin label that could be doubly incorporated into T4 Lysozyme (T4L) by site-directed spin labeling with efficiency up to 50% per cysteine. The rigidity of the fused ring structure and absence of mobile methyl groups increase the spin echo dephasing time (Tm) at temperatures above 80 K. This enables DEER measurements of distances >4 nm in DZD-labeled T4L in glycerol/water at temperatures up to 150 K with increased sensitivity compared to that of a common spin label such as MTSL. Addition of ß-cyclodextrin reduces the rotational correlation time of the label, slightly increases Tm, and most importantly, narrows (and slightly lengthens) the interspin distance distributions. The distance distributions are in good agreement with simulated distance distributions obtained by rotamer libraries. These results provide a foundation for developing supramolecular recognition to facilitate long-distance DEER measurements at near physiological temperatures.

Counterion influence on dynamic spin properties in a V(iv) complex.

Using transition metal ions for spin-based applications, such as electron paramagnetic resonance imaging (EPRI) or quantum computation, requires a clear understanding of how local chemistry influences spin properties. Herein we report a series of four ionic complexes to provide the first systematic study of one aspect of local chemistry on the V(iv) spin - the counterion. To do so, the four complexes (Et3NH)2[V(C6H4O2)3] (1), (n-Bu3NH)2[V(C6H4O2)3] (2), (n-Hex3NH)2[V(C6H4O2)3] (3), and (n-Oct3NH)2[V(C6H4O2)3] (4) were probed by EPR spectroscopy in solid state and solution. Room temperature, solution X-band (ca. 9.8 GHz) continuous-wave electron paramagnetic resonance (CW-EPR) spectroscopy revealed an increasing linewidth with larger cations, likely a counterion-controlled tumbling in solution via ion pairing. In the solid state, variable-temperature (5-180 K) X-band (ca. 9.4 GHz) pulsed EPR studies of 1-4 in o-terphenyl glass demonstrated no effect on spin-lattice relaxation times (T 1), indicating little role for the counterion on this parameter. However, the phase memory time (T m) of 1 below 100 K is markedly smaller than those of 2-4. This result is counterintuitive, as 2-4 are relatively richer in 1H nuclear spin, hence, expected to have shorter T m. Thus, these data suggest an important role for counterion methyl groups on T m, and moreover provide the first instance of a lengthening T m with increasing nuclear spin quantity on a molecule.

Structural insights for vanadium catecholates and iron­sulfur clusters obtained from multiple data analysis methods applied to electron spin relaxation data.

Electron paramagnetic resonance (EPR) inversion recovery curves for vanadium catecholates and iron­sulfur clusters were analyzed with three models: the sum of two exponentials, a stretched exponential, and a model-free distribution of exponentials (UPEN). For all data sets studied fits with a stretched exponential were statistically indistinguishable from the sum of two exponentials, and were significantly better than for single exponentials. UPEN provides insights into the structures of the distributions. For a vanadium(IV) tris catecholate the distribution of relaxation rates calculated with UPEN shows the contribution from spectral diffusion at low temperatures. The energy of the local mode for this complex, found from the temperature dependence of the spin lattice relaxation, is consistent with values expected for a metal-ligand vibration. For the [2Fe-2S]+ cluster in pyruvate formate lyase activating enzyme (PFL-AE) the small stretched exponential ß values (0.3) at low temperature and the distributions calculated with UPEN reflect the contribution from a second rapidly relaxing species that could be difficult to detect by continuous wave EPR. The distributions in 1/T1 for the [4Fe-4S]+ clusters in Mycofactocin maturase were about a factor of four wider than for the three other systems studied. The very broad distribution of relaxation rates may be due to protein mobility and distributions in electronic energies and local environments for the clusters. UPEN provides insight into several situations that can result in low values of stretch parameter ß including contributions from spectral diffusion, overlapping signals from distinguishable clusters, or very wide distributions.

Electron paramagnetic resonance of a 10B-containing heterocyclic radical.

Electron paramagnetic resonance measurements for a 10B-containing heterocyclic phenanthrenedione radical, (C6F5)2B(O2C14H8), were made at X-band in 9:1 toluene:dichloromethane from 10 to 293 K and in toluene from 180 to 293 K. In well-deoxygenated 0.1 mM toluene solution at room temperature hyperfine couplings to 10B, four pairs of protons and five pairs of fluorines contribute to a continuous wave spectrum with many resolved lines. Hyperfine couplings were adjusted to provide the best fit for spectra of the radical enriched in 10B and the analogous radical synthesized with 10,11B in natural abundance, resulting in small refinements of the hyperfine coupling constants previously reported for the natural abundance sample. Electron spin relaxation rates at temperatures between 15 and 293 K were similar for samples containing 10B and natural isotope abundance. Analysis of electron spin echo envelope modulation and hyperfine correlation spectroscopy data at 80 K found Axx = -7.5 ±â€¯0.3, Ayy = -8.5 ±â€¯0.3, and Azz = -10.8 ±â€¯0.3 MHz for 11B, which indicates small spin density on the boron. The spin echo and hyperfine spectroscopy data for the 10B -containing radical are consistent with the factor of 2.99 smaller hyperfine values for 10B than for 11B.