The reaction catalyzed by E. coli ribonucleotide reductase (RNR) composed of α and β subunits that form an active α2β2 complex is a paradigm for proton-coupled electron transfer (PCET) processes in biological transformations. β2 contains the diferric tyrosyl radical (Y·) cofactor that initiates radical transfer (RT) over 35 Å via a specific pathway of amino acids (Y· ⇆ [W] ⇆ Y in β2 to Y ⇆ Y ⇆ C in α2). Experimental evidence exists for colinear and orthogonal PCET in α2 and β2, respectively. No mechanistic model yet exists for the PCET across the subunit (α/β) interface. Here, we report unique EPR spectroscopic features of Y·-β, the pathway intermediate generated by the reaction of 2,3,5-FY·-β2/CDP/ATP with wt-α2, YF-α2, or YF-α2. High field EPR (94 and 263 GHz) reveals a dramatically perturbed g tensor. [H] and [H]-ENDOR reveal two exchangeable H bonds to Y·: 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 (r ∼1.8-1.9 Å) are required to reproduce the g value of Y· (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 Y· 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 Y oxidation.

A new conformation of the E. coli RNR pathway residue 731 was trapped during long-range radical transfer across the αβ subunit interface.

Fullerene C 60 and its derivatives are widely used in molecular electronics, photovoltaics, and battery materials, because of their exceptional suitability as electron acceptors. In this context, single-electron transfer on C 60 generates the C 60 • − radical anion. However, the short lifetime of free C 60• − hampers its investigation and application. In this work, we dramatically stabilize the usually short-lived C 60• − species within a self-assembled M 2 L 4 coordination cage consisting of a triptycene-based ligand and Pd(II) cations. The electron-deficient cage strongly binds C 60 by providing a curved inner π-surface complementary to the fullerene's globular shape. Cyclic voltammetry revealed a positive potential shift for the first reduction of encapsulated C 60 , which is indicative of a strong interaction between confined C 60• − and the cationic cage. Photochemical one-electron reduction with 1benzyl-1,4-dihydronicotinamide allows selective and quantitative conversion of the confined C 60 molecule in millimolar acetonitrile solution at room temperature. Radical generation was confirmed by nuclear magnetic resonance, electron paramagnetic resonance, ultraviolet−visible−near-infrared spectroscopy and electrospray ionization mass spectrometry. The lifetime of C 60• − within the cage was determined to be so large that it could still be detected after one month under an inert atmosphere.

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The homo- and heterocoupling of molecular terminal iridium(iv) and iridium(v) nitrides is examined. The experimental coupling rates are discussed based on a computational analysis of the transition states.

DNA G-quadruplexes show a pronounced tendency to form higher-order structures, such as p-stacked dimers and aggregates with aromatic binding partners. Reliable methods for determining the structure of these non-covalent adducts are scarce. Here, we use artificial square-planar Cu(pyridine) 4 complexes, covalently incorporated into tetramolecular Gquadruplexes, as rigid spin labels for detecting dimeric structures and measuring intermolecular Cu 2+-Cu 2+ distances via pulsed dipolar EPR spectroscopy. A series of G-quadruplex dimers of different spatial dimensions, formed in tail-totail or head-to-head stacking mode, were unambiguously distinguished. Measured distances are in full agreement with results of molecular dynamics simulations. Furthermore, intercalation of two well-known G-quadruplex binders, PIPER and telomestatin, into G-quadruplex dimers resulting in sandwich complexes was investigated, and previously unknown binding modes were discovered. Additionally, we present evidence that free G-tetrads also intercalate into dimers. Our transition metal labeling approach, combined with pulsed EPR spectroscopy, opens new possibilities for examining structures of non-covalent DNA aggregates.

Singlet vinylidenes (R 2 C=C:) are proposed as intermediates in a series of organic reactions, and very few have been studied by matrix isolation or gas-phase spectroscopy. Triplet vinylidenes, however, featuring two unpaired electrons at a monosubstituted carbon atom are thus far only predicted as electronically excited-state species and represent an unexplored class of carbon-centered diradicals. We report the photochemical generation and low-temperature EPR/ENDOR characterization of the first ground-state high-spin (triplet) vinylidene. The zero-field splitting parameters ( D = 0.377 cm –1 and | E| / D = 0.028) were determined, and the 13 C hyperfine coupling tensor was obtained by 13 C-ENDOR measurements. Most strikingly, the isotropic 13 C hyperfine coupling constant (50 MHz) is far smaller than the characteristic values of triplet carbenes, demonstrating a unique electronic structure which is supported by quantum chemical calculations.

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 ΔE°’ 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 RNR, which when incubated with β2/CDP/ATP generates the D6-NH2Y731•-α2/β2 complex. By multi-frequency EPR (35, 94 and 263 GHz) and 34 GHz 1H ENDOR spectroscopies, we determined the hyperfine coupling (hfc) constants of the amino protons that establishes 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 NH2YRS/tRNA pair was determined by a generally useful LC-MS method. This information is essential to the usefulness of this NH2Y probe to study any protein of interest and is employed to address our previously reported activity associated with NH2Y-substituted RNRs.