Ribonucleotide Reductases: Structure, Chemistry, and Metabolism Suggest New Therapeutic Targets.

Ribonucleotide reductases (RNRs) catalyze the de novo conversion of nucleotides to deoxynucleotides in all organisms, controlling their relative ratios and abundance. In doing so, they play an important role in fidelity of DNA replication and repair. RNRs' central role in nucleic acid metabolism has resulted in five therapeutics that inhibit human RNRs. In this review, we discuss the structural, dynamic, and mechanistic aspects of RNR activity and regulation, primarily for the human and Escherichia coli class Ia enzymes. The unusual radical-based organic chemistry of nucleotide reduction, the inorganic chemistry of the essential metallo-cofactor biosynthesis/maintenance, the transport of a radical over a long distance, and the dynamics of subunit interactions all present distinct entry points toward RNR inhibition that are relevant for drug discovery. We describe the current mechanistic understanding of small molecules that target different elements of RNR function, including downstream pathways that lead to cell cytotoxicity. We conclude by summarizing novel and emergent RNR targeting motifs for cancer and antibiotic therapeutics.

Statistical analysis of ENDOR spectra.

Electron-nuclear double resonance (ENDOR) measures the hyperfine interaction of magnetic nuclei with paramagnetic centers and is hence a powerful tool for spectroscopic investigations extending from biophysics to material science. Progress in microwave technology and the recent availability of commercial electron paramagnetic resonance (EPR) spectrometers up to an electron Larmor frequency of 263 GHz now open the opportunity for a more quantitative spectral analysis. Using representative spectra of a prototype amino acid radical in a biologically relevant enzyme, the [Formula: see text] in Escherichia coli ribonucleotide reductase, we developed a statistical model for ENDOR data and conducted statistical inference on the spectra including uncertainty estimation and hypothesis testing. Our approach in conjunction with 1H/2H isotopic labeling of [Formula: see text] in the protein unambiguously established new unexpected spectral contributions. Density functional theory (DFT) calculations and ENDOR spectral simulations indicated that these features result from the beta-methylene hyperfine coupling and are caused by a distribution of molecular conformations, likely important for the biological function of this essential radical. The results demonstrate that model-based statistical analysis in combination with state-of-the-art spectroscopy accesses information hitherto beyond standard approaches.

Rational Design of a Phosphorus-Centered Disbiradical.

Phosphorus-centered disbiradicals, in which the radical sites exist as individual spin doublets with weak spin-spin interaction have not been known so far. Starting from monoradicals of the type [⋅P(µ-NTer)2 P-R], we have now succeeded in linking two such monoradical phosphorus centers by appropriate choice of a linker. To this end, biradical [⋅P(µ-NTer)2 P⋅] (1) was treated with 1,6-dibromohexane, affording the brominated species {Br[P(µ-NTer)]2 }2 C6 H12 (3). Subsequent reduction with KC8 led to the formation of the disbiradical {⋅[P(µ-NTer)]2 }2 C6 H12 (4) featuring a large distance between the radical phosphorus sites in the solid state and formally the highest biradical character observed in a P-centered biradical so far, approaching 100 %. EPR spectroscopy revealed a three-line signal in solution with a considerably larger exchange interaction than would be expected from the molecular structure of the single crystal. Quantum chemical calculations revealed a highly dynamic conformational space; thus, the two radical sites can approach each other with a much smaller distance in solution. Further reduction of 4 resulted in the formation of a potassium salt featuring the first structurally characterized P-centered distonic radical anion (5- ). Moreover, 4 could be used in small molecule activation.

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.

Large 31P-NMR enhancements in liquid state dynamic nuclear polarization through radical/target molecule non-covalent interaction.

Dynamic nuclear polarization (DNP) is a method to enhance the low sensitivity of nuclear magnetic resonance (NMR) via spin polarization transfer from electron spins to nuclear spins. In the liquid state, this process is mediated by fast modulations of the electron-nuclear hyperfine coupling and its efficiency depends strongly on the applied magnetic field. A peculiar case study is triphenylphosphine (PPh3) dissolved in benzene and doped with BDPA radical because it gives 31P-NMR signal enhancements of two orders of magnitude up to a magnetic field of 14.1 T. Here we show that the large 31P enhancements of BDPA/PPh3 in benzene at 1.2 T (i) decrease when the moieties are dissolved in other organic solvents, (ii) are strongly reduced when using a nitroxide radical, and (iii) vanish with pentavalent 31P triphenylphosphine oxide. Those experimental observations are rationalized with numerical calculations based on density functional theory that show the tendency of BDPA and PPh3 to form a weak complex via non-covalent interaction that leads to large hyperfine couplings to 31P (ΔAiso ≥ 13 MHz). This mechanism is hampered in other investigated systems. The case study of 31P-DNP in PPh3 is an important example that extends the current understanding of DNP in the liquids state: non-covalent interactions between radical and target can be particularly effective to obtain large NMR signal enhancements.

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.

Benchmark Test and Guidelines for DEER/PELDOR Experiments on Nitroxide-Labeled Biomolecules.

Distance distribution information obtained by pulsed dipolar EPR spectroscopy provides an important contribution to many studies in structural biology. Increasingly, such information is used in integrative structural modeling, where it delivers unique restraints on the width of conformational ensembles. In order to ensure reliability of the structural models and of biological conclusions, we herein define quality standards for sample preparation and characterization, for measurements of distributed dipole-dipole couplings between paramagnetic labels, for conversion of the primary time-domain data into distance distributions, for interpreting these distributions, and for reporting results. These guidelines are substantiated by a multi-laboratory benchmark study and by analysis of data sets with known distance distribution ground truth. The study and the guidelines focus on proteins labeled with nitroxides and on double electron-electron resonance (DEER aka PELDOR) measurements and provide suggestions on how to proceed analogously in other cases.

Studies of transmembrane peptides by pulse dipolar spectroscopy with semi-rigid TOPP spin labels.

Electron paramagnetic resonance (EPR)-based pulsed dipolar spectroscopy measures the dipolar interaction between paramagnetic centers that are separated by distances in the range of about 1.5-10 nm. Its application to transmembrane (TM) peptides in combination with modern spin labelling techniques provides a valuable tool to study peptide-to-lipid interactions at a molecular level, which permits access to key parameters characterizing the structural adaptation of model peptides incorporated in natural membranes. In this mini-review, we summarize our approach for distance and orientation measurements in lipid environment using novel semi-rigid TOPP [4-(3,3,5,5-tetramethyl-2,6-dioxo-4-oxylpiperazin-1-yl)-L-phenylglycine] labels specifically designed for incorporation in TM peptides. TOPP labels can report single peak distance distributions with sub-angstrom resolution, thus offering new capabilities for a variety of TM peptide investigations, such as monitoring of various helix conformations or measuring of tilt angles in membranes.

Spin density localization and accessibility of organic radicals affect liquid-state DNP efficiency.

We report a large variation in liquid DNP performance of up to a factor of about five in coupling factor among organic radicals commonly used as polarizing agents. A comparative study of 1H and 13C DNP in model systems shows the impact of the spin density distribution and accessibility of the radical site by the target molecule.

Measurement of Angstrom to Nanometer Molecular Distances with 19 F Nuclear Spins by EPR/ENDOR Spectroscopy.

Spectroscopic and biophysical methods for structural determination at atomic resolution are fundamental in studies of biological function. Here we introduce an approach to measure molecular distances in bio-macromolecules using 19 F nuclear spins and nitroxide radicals in combination with high-frequency (94 GHz/3.4 T) electron-nuclear double resonance (ENDOR). The small size and large gyromagnetic ratio of the 19 F label enables to access distances up to about 1.5 nm with an accuracy of 0.1-1 Å. The experiment is not limited by the size of the bio-macromolecule. Performance is illustrated on synthesized fluorinated model compounds as well as spin-labelled RNA duplexes. The results demonstrate that our simple but strategic spin-labelling procedure combined with state-of-the-art spectroscopy accesses a distance range crucial to elucidate active sites of nucleic acids or proteins in the solution state.

Semi-Rigid Nitroxide Spin Label for Long-Range EPR Distance Measurements of Lipid Bilayer Embedded ß-Peptides.

ß-Peptides are an interesting new class of transmembrane model peptides based on their conformationally stable and well-defined secondary structures. Herein, we present the synthesis of the paramagnetic ß-amino acid ß3 -hTOPP (4-(3,3,5,5-tetramethyl-2,6-dioxo-4-oxylpiperazin-1-yl)-d-ß3 -homophenylglycine) that enables investigations of ß-peptides by EPR spectroscopy. This amino acid adds to the, to date, sparse number of ß-peptide spin labels. Its performance was evaluated by investigating the helical turn of a 314 -helical transmembrane model ß-peptide. Nanometer distances between two incorporated ß3 -hTOPP labels in different environments were measured by using pulsed electron/electron double resonance (PELDOR/DEER) spectroscopy. Due to the semi-rigid conformational design, the label delivers reliable distances and sharp (one-peak) distance distributions even in the lipid bilayer. The results indicate that the investigated ß-peptide folds into a 3.2514 helix and maintains this conformation in the lipid bilayer.

Dynamic Nuclear Polarization of 13 C Nuclei in the Liquid State over a 10†Tesla Field Range.

Nuclear magnetic resonance (NMR) techniques play an essential role in natural science and medicine. In spite of the tremendous utility associated with the small energies detected, the most severe limitation is the low signal-to-noise ratio. Dynamic nuclear polarization (DNP), a technique based on transfer of polarization from electron to nuclear spins, has emerged as a tool to enhance sensitivity of NMR. However, the approach in liquids still faces several challenges. Herein we report the observation of room-temperature, liquid DNP 13 C signal enhancements in organic small molecules as high as 600 at 9.4 Tesla and 800 at 1.2 Tesla. A mechanistic investigation of the 13 C-DNP field dependence shows that DNP efficiency is raised by proper choice of the polarizing agent (paramagnetic center) and by halogen atoms as mediators of scalar hyperfine interaction. Observation of sizable DNP of 13 CH2 and 13 CH3 groups in organic molecules at 9.4 T opens perspective for a broader application of this method.

Properties of Site-Specifically Incorporated 3-Aminotyrosine in Proteins To Study Redox-Active Tyrosines: Escherichia coli Ribonucleotide Reductase as a Paradigm.

3-Aminotyrosine (NH2Y) has been a useful probe to study the role of redox active tyrosines in enzymes. This report describes properties of NH2Y of key importance for its application in mechanistic studies. By combining the tRNA/NH2Y-RS suppression technology with a model protein tailored for amino acid redox studies (α3X, X = NH2Y), the formal reduction potential of NH2Y32(O•/OH) ( E°' = 395 ± 7 mV at pH 7.08 ± 0.05) could be determined using protein film voltammetry. We find that the Δ E°' between NH2Y32(O•/OH) and Y32(O•/OH) when measured under reversible conditions is ∼300-400 mV larger than earlier estimates based on irreversible voltammograms obtained on aqueous NH2Y and Y. We have also generated D6-NH2Y731-α2 of ribonucleotide reductase (RNR), which when incubated with ß2/CDP/ATP generates the D6-NH2Y731•-α2/ß2 complex. By multifrequency electron paramagnetic resonance (35, 94, and 263 GHz) and 34 GHz 1H ENDOR spectroscopies, we determined the hyperfine coupling (hfc) constants of the amino protons that establish RNH2• planarity and thus minimal perturbation of the reduction potential by the protein environment. The amount of Y in the isolated NH2Y-RNR incorporated by infidelity of the tRNA/NH2Y-RS pair was determined by a generally useful LC-MS method. This information is essential to the utility of this NH2Y probe to study any protein of interest and is employed to address our previously reported activity associated with NH2Y-substituted RNRs.

Spectroscopic Evidence for a H Bond Network at Y356 Located at the Subunit Interface of Active E. coli Ribonucleotide Reductase.

The reaction catalyzed by E. coli ribonucleotide reductase (RNR) composed of α and ß subunits that form an active α2ß2 complex is a paradigm for proton-coupled electron transfer (PCET) processes in biological transformations. ß2 contains the diferric tyrosyl radical (Y122·) cofactor that initiates radical transfer (RT) over 35 Å via a specific pathway of amino acids (Y122· ⇆ [W48] ⇆ Y356 in ß2 to Y731 ⇆ Y730 ⇆ C439 in α2). Experimental evidence exists for colinear and orthogonal PCET in α2 and ß2, respectively. No mechanistic model yet exists for the PCET across the subunit (α/ß) interface. Here, we report unique EPR spectroscopic features of Y356·-ß, the pathway intermediate generated by the reaction of 2,3,5-F3Y122·-ß2/CDP/ATP with wt-α2, Y731F-α2, or Y730F-α2. High field EPR (94 and 263 GHz) reveals a dramatically perturbed g tensor. [1H] and [2H]-ENDOR reveal two exchangeable H bonds to Y356·: a moderate one almost in-plane with the π-system and a weak one. DFT calculation on small models of Y· indicates that two in-plane, moderate H bonds (rO-H ∼1.8-1.9 Å) are required to reproduce the gx value of Y356· (wt-α2). The results are consistent with a model, in which a cluster of two, almost symmetrically oriented, water molecules provide the two moderate H bonds to Y356· that likely form a hydrogen bond network of water molecules involved in either the reversible PCET across the subunit interface or in H+ release to the solvent during Y356 oxidation.

Photo-induced radical polarization and liquid-state dynamic nuclear polarization using fullerene nitroxide derivatives.

We report on radical polarization and optically-driven liquid DNP using nitroxide radicals functionalized by photoexcitable fullerene derivatives. Pulse laser excitation of the fullerene moiety leads to transient nitroxide radical polarization that is one order of magnitude larger than that at the Boltzmann equilibrium. The life time of the radical polarization increases with the size of the fullerene derivative and is correlated with the electronic spin-lattice relaxation time T1e. Overhauser NMR signal enhancements of toluene solvent protons were observed under steady-state illumination, which replaced microwave irradiation.

Pulse EPR Measurements of Intramolecular Distances in a TOPP-Labeled Transmembrane Peptide in Lipids.

We present the performance of nanometer-range pulse electron paramagnetic resonance distance measurements (pulsed electron-electron double resonance/double electron-electron resonance, PELDOR/DEER) on a transmembrane WALP24 peptide labeled with the semirigid unnatural amino acid 4-(3,3,5,5-tetra-methyl-2,6-dioxo-4-oxylpiperazin-1-yl)-l-phenylglycine (TOPP). Distances reported by the TOPP label are compared to the ones reported by the more standard MTSSL spin label, commonly employed in protein studies. Using high-power pulse electron paramagnetic resonance spectroscopy at Q-band frequencies (34 GHz), we show that in contrast to MTSSL, our label reports one-peak, sharp (Δr ≤ 0.4 nm) intramolecular distances. Orientational selectivity is not observed. When spin-labeled WALP24 was inserted in two representative lipid bilayers with different bilayer thickness, i.e., DMPC and POPC, the intramolecular distance reported by TOPP did not change with the bilayer environment. In contrast, the distance measured with MTSSL was strongly affected by the hydrophobic thickness of the lipid. The results demonstrate that the TOPP label is well suited to study the intrinsic structure of peptides immersed in lipids.

Hydrogen bond network between amino acid radical intermediates on the proton-coupled electron transfer pathway of E. coli α2 ribonucleotide reductase.

Ribonucleotide reductases (RNRs) catalyze the conversion of ribonucleotides to deoxyribonucleotides in all organisms. In all Class Ia RNRs, initiation of nucleotide diphosphate (NDP) reduction requires a reversible oxidation over 35 Å by a tyrosyl radical (Y122•, Escherichia coli) in subunit ß of a cysteine (C439) in the active site of subunit α. This radical transfer (RT) occurs by a specific pathway involving redox active tyrosines (Y122 ⇆ Y356 in ß to Y731 ⇆ Y730 ⇆ C439 in α); each oxidation necessitates loss of a proton coupled to loss of an electron (PCET). To study these steps, 3-aminotyrosine was site-specifically incorporated in place of Y356-ß, Y731- and Y730-α, and each protein was incubated with the appropriate second subunit ß(α), CDP and effector ATP to trap an amino tyrosyl radical (NH2Y•) in the active α2ß2 complex. High-frequency (263 GHz) pulse electron paramagnetic resonance (EPR) of the NH2Y•s reported the gx values with unprecedented resolution and revealed strong electrostatic effects caused by the protein environment. (2)H electron-nuclear double resonance (ENDOR) spectroscopy accompanied by quantum chemical calculations provided spectroscopic evidence for hydrogen bond interactions at the radical sites, i.e., two exchangeable H bonds to NH2Y730•, one to NH2Y731• and none to NH2Y356•. Similar experiments with double mutants α-NH2Y730/C439A and α-NH2Y731/Y730F allowed assignment of the H bonding partner(s) to a pathway residue(s) providing direct evidence for colinear PCET within α. The implications of these observations for the PCET process within α and at the interface are discussed.

Cross-Polarization Electron-Nuclear Double Resonance Spectroscopy.

Magnetic nuclei in the proximity of a paramagnetic center can be polarized through electron-nuclear cross-polarization and detected in electron-nuclear double resonance (ENDOR) spectroscopy. This principle is demonstrated in a single-crystal model sample as well as on a protein, the ß2 subunit of E.coli ribonucleotide reductase (RNR), which contains an essential tyrosyl radical. ENDOR is a fundamental technique to detect magnetic nuclei coupled to paramagnetic centers. It is widely employed in biological and materials sciences. Despite its utility, its sensitivity in real samples is about one to two orders of magnitude lower than conventional electron paramagnetic resonance, thus restricting its application potential. Herein, we report the performance of a recently introduced concept to polarize nuclear spins and detect their ENDOR spectrum, which is based on electron-nuclear cross polarization (eNCP). A single-crystal study permits us to disentangle eNCP conditions and CP-ENDOR intensities, providing the experimental foundation in agreement with the theoretical prediction. The CP-ENDOR performance on a real protein sample is best demonstrated with the spectra of the essential tyrosyl radical in the ß2 subunit of E.coli RNR.

A high saturation factor in Overhauser DNP with nitroxide derivatives: the role of (14)N nuclear spin relaxation.

Overhauser DNP enhancements of toluene were measured at a magnetic field of 0.35 Tesla in a series of chemically functionalized nitroxide radicals. We observe that the enhancements increase systematically with polarizer size and rotational correlation time. Examination of the saturation factor of (14)N nitroxides by pulsed ELDOR spectroscopy led to a quantitative interpretation of the enhancements, for which the saturation factor increases up to almost unity due to enhanced nuclear ((14)N) relaxation in the nitroxide radical. The observation has a direct impact on the choice of optimum DNP polarizers in liquids.