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.

The E. coli ribonucleotide reductase (RNR), a paradigm for class Ia enzymes including human RNR, catalyzes the biosynthesis of DNA building blocks and requires a di‐iron tyrosyl radical (Y122.) cofactor for activity. The knowledge on the in vitro Y122. structure and its radical distribution within the β2 subunit has accumulated over the years; yet little information exists on the in vivo Y122.. Here, we characterize this essential radical in whole cells. Multi‐frequency EPR and electron‐nuclear double resonance (ENDOR) demonstrate that the structure and electrostatic environment of Y122. are identical under in vivo and in vitro conditions. Pulsed dipolar EPR experiments shed light on a distinct in vivo Y122. per β2 distribution, supporting the key role of Y. concentrations in regulating RNR activity. Additionally, we spectroscopically verify the generation of an unnatural amino acid radical, F3Y122., in whole cells, providing a crucial step towards unique insights into the RNR catalysis under physiological conditions.

In biological cells, osmolytes appear as complex mixtures with variable compositions, depending on the particular environmental conditions of the organism. Based on various spectroscopic, thermodynamic and small-angle scattering data, we explored the effect of two different natural osmolyte mixtures, which are found in shallow-water and deep-sea shrimps, on the temperature and pressure stability of a typical monomeric protein, RNase A. Both natural osmolyte mixtures stabilize the protein against thermal and pressure denaturation. This effect seems to be mainly caused by the major osmolyte components of the osmolyte mixtures, i.e. by glycine and trimethylamine-N-oxide (TMAO), respectively. A minor compaction of the structure, in particular in the unfolded state, seems to be largely due to TMAO. Differences in thermodynamic properties observed for glycine and TMAO, and hence also for the two osmolyte mixtures, are most likely due to different solvation properties and interactions with the protein. Different from TMAO, glycine seems to interact with the amino acid side chains and/or the backbone of the protein, thus competing with hydration water and leading to a less hydrated protein surface.

The E. coli ribonucleotide reductase (RNR), a paradigm for class Ia enzymes including human RNR, catalyzes the biosynthesis of DNA building blocks and requires a di‐iron tyrosyl radical (Y122.) cofactor for activity. The knowledge on the in vitro Y122. structure and its radical distribution within the β2 subunit has accumulated over the years; yet little information exists on the in vivo Y122.. Here, we characterize this essential radical in whole cells. Multi‐frequency EPR and electron‐nuclear double resonance (ENDOR) demonstrate that the structure and electrostatic environment of Y122. are identical under in vivo and in vitro conditions. Pulsed dipolar EPR experiments shed light on a distinct in vivo Y122. per β2 distribution, supporting the key role of Y. concentrations in regulating RNR activity. Additionally, we spectroscopically verify the generation of an unnatural amino acid radical, F3Y122., in whole cells, providing a crucial step towards unique insights into the RNR catalysis under physiological conditions.

The structure and regulation of the tyrosyl radical in ribonucleotide reductase in whole E. coli cells was investigated by using advanced EPR spectroscopy. This radical starts the biosynthesis of DNA building blocks, and is thus involved in essential processes in life. The observations reported by Müge Kasanmascheff et al. in their Research Article on page 19155 were surprising and exciting because the findings indicated a distinct radical distribution within the cells, pointing to a complex regulation mechanism of this fundamental enzyme in its native environment.