Ribonucleotide reductases (RNRs) catalyze the conversion of ribonucleotide (RNA) to deoxyribonucleotide (DNA) building blocks initiated by a long-range (>30 Å) proton-coupled electron transfer (PCET) by mechanistic principles that remain much debated. By combining multiscale quantum and classical simulations with directed mutagenesis, X-ray crystallography, and vibrational and electron paramagnetic resonance spectroscopy, we elucidate here the molecular principles underlying how metal-free RNRs initiate the long-range PCET process by creating a highly stable 3,4-dihydroxyphenylalanine (DOPA) initiator radical. We show that DOPA• is redox-tuned by a low-barrier hydrogen bond (LBHB), with a delocalized proton that provides the catalytic power for the ribonucleotide reduction. We find that the LBHB couples to an extended hydrogen-bonded network, with distant mutations resulting in the loss of radical formation, and providing key molecular insight into the long-range radical transport mechanism in RNRs. On a general level, our findings support the direct involvement of LBHB in protein chemistry and the importance of quantum effects in enzyme catalysis.

Class I ribonucleotide reductases (RNRs) convert ribonucleotides into deoxyribonucleotides under oxic conditions. The R2 subunit provides a radical required for catalysis conducted by the R1 subunit. In most R2s the radical is generated on a tyrosine via oxidation by an adjacent metal site. The discovery of a metal-free R2 defined the new RNR subclass Ie. In R2e, three of the otherwise strictly conserved metal-binding glutamates in the active site are substituted. Two variants have been found, VPK and QSK. To date, the VPK version has been the focus of biochemical characterization. Here we characterize a QSK variant of R2e. We analyse the organismal distribution of the two R2e versions and find dozens of organisms relying solely on the QSK RNR for deoxyribonucleotide production. We demonstrate that the R2e_QSK of the human pathogen Gardnerella vaginalis (Bifidobacterium vaginale) modifies the active site-adjacent tyrosine to DOPA. The amount of modified protein is shown to be dependent on coexpression with the other proteins encoded in the RNR operon. The DOPA containing R2e_QSK can support ribonucleotide reduction in vitro while the unmodified protein cannot. Finally, we determined the first structures of R2e_QSK in the unmodified and DOPA states.

Understanding the structure of an active pharmaceutical ingredient is essential for gaining insights into its physicochemical properties. Sodium valproate, one of the most effective antiepileptic drugs, was first approved for medical use in 1967. However, the structure of its anhydrous form has remained unresolved. This is because it was difficult to grow crystals of sufficient size for single-crystal X-ray diffraction (SCXRD). Although 3D electron diffraction (3D ED) can be used for studying crystals that are too small for SCXRD, the crystals of anhydrous sodium valproate are extremely sensitive to both humidity and electron beams. They degrade quickly both in air and under an electron beam at room temperature. In this study, we developed a glovebox-assisted cryo-transfer workflow for the preparation of EM grids in a protected atmosphere to overcome the current challenges for studying air- and beam-sensitive samples using 3D ED. Using this technique, we successfully determined the structure of anhydrous sodium valproate, revealing the formation of Na-valproate polyhedral chains. Our results provide a robust framework for the 3D ED analysis of air-sensitive crystals, greatly enhancing its utility across various scientific disciplines.

Aerobic ribonucleotide reductases (RNRs) initiate synthesis of DNA building blocks by generating a free radical within the R2 subunit; the radical is subsequently shuttled to the catalytic R1 subunit through proton-coupled electron transfer (PCET). We present a high-resolution room temperature structure of the class Ie R2 protein radical captured by x-ray free electron laser serial femtosecond crystallography. The structure reveals conformational reorganization to shield the radical and connect it to the translocation path, with structural changes propagating to the surface where the protein interacts with the catalytic R1 subunit. Restructuring of the hydrogen bond network, including a notably short O–O interaction of 2.41 angstroms, likely tunes and gates the radical during PCET. These structural results help explain radical handling and mobilization in RNR and have general implications for radical transfer in proteins.