Vivek Srinivas

Carbon monoxide dehydrogenases (CODHs) catalyse the reversible oxidation of CO to CO ₂ and play central roles in microbial carbon metabolism. While well‐characterised CODHs from different phylogenetic backgrounds exhibit high bidirectional activity, the enigmatic clade B remains functionally uncharacterised. Here, we present the first structural and biochemical characterisation of a clade B CODH from Ruminococcus flavefaciens ( Rf CODH). It reveals striking divergence from canonical enzymes. A new anaerobic cryo‐EM workflow was developed, carried out entirely under anoxic conditions by manual blotting and plunge freezing. It resulted in a 2.53 Å Rf CODH structure. The structure adopts the typical CODH fold, but exhibits blocked gas channels, a compromised proton transfer pathway and disrupted cofactor coordination. This provides a structural rationale for Rf CODH's severely attenuated CO oxidation activity (13 mU/mg vs. 900 U/mg for the well‐studied Ch CODH‐II). EPR spectroscopy reveals unique oxidised C‐cluster states not previously characterised in CODHs. Mirror tree analysis hints to co‐evolution between clade B CODHs and associated ABC transporter substrate‐binding proteins, suggesting these enzymes function in metabolism of substrates imported via the ABC transporter module. All findings indicate evolutionary repurposing of the CODH scaffold for alternative physiological functions.

Carbon monoxide dehydrogenases (CODHs) catalyse the reversible oxidation of CO to CO ₂ and play central roles in microbial carbon metabolism. While well‐characterised CODHs from different phylogenetic backgrounds exhibit high bidirectional activity, the enigmatic clade B remains functionally uncharacterised. Here, we present the first structural and biochemical characterisation of a clade B CODH from Ruminococcus flavefaciens ( Rf CODH). It reveals striking divergence from canonical enzymes. A new anaerobic cryo‐EM workflow was developed, carried out entirely under anoxic conditions by manual blotting and plunge freezing. It resulted in a 2.53 Å Rf CODH structure. The structure adopts the typical CODH fold, but exhibits blocked gas channels, a compromised proton transfer pathway and disrupted cofactor coordination. This provides a structural rationale for Rf CODH's severely attenuated CO oxidation activity (13 mU/mg vs. 900 U/mg for the well‐studied Ch CODH‐II). EPR spectroscopy reveals unique oxidised C‐cluster states not previously characterised in CODHs. Mirror tree analysis hints to co‐evolution between clade B CODHs and associated ABC transporter substrate‐binding proteins, suggesting these enzymes function in metabolism of substrates imported via the ABC transporter module. All findings indicate evolutionary repurposing of the CODH scaffold for alternative physiological functions.

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.

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 combing 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 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. Significance Statement: Ribonucleotide reductases (RNRs) are ancient enzymes responsible for the synthesis of deoxyribonucleotides from ribonucleotides. RNRs catalyze this reaction via a long-range proton-coupled electron transfer (PCET) process, involving the formation of a stable protein radical. Yet, despite decades of detailed structural, biochemical, spectroscopic and computational studies, the mechanistic principles of this process remain unclear and much debated. Here, we show that metal-free RNRs power the reduction of RNA building blocks by a highly stable organic DOPA initiator radical, arising from a unique low-barrier hydrogen bonding (LBHB) network that enables the radical transport by strong redox-tuning effects. Our findings reveal mechanistic principles underlying the elusive PCET reactions of metal-free RNRs, and provide evidence for the involvement of quantum effects in enzyme catalysis.

Carbon monoxide dehydrogenases (CODHs) catalyse the reversible oxidation of CO to CO₂ and play central roles in microbial carbon metabolism. While well-characterised CODHs from different phylogenetic backgrounds exhibit high bidirectional activity, the enigmatic clade B remains functionally uncharacterised. Here we present the first structural and functional characterisation of a clade B CODH from Ruminococcus flavefaciens ( Rf CODH). It reveals striking divergence from canonical enzymes. A new anaerobic cryo-EM workflow was developed, carried out entirely under anoxic conditions by manual blotting and plunge freezing. It resulted in a 2.53 Å Rf CODH structure. The structure adopts the typical CODH fold, but exhibits blocked gas channels, a compromised proton transfer pathway and disrupted cofactor coordination. This provides a structural rationale for Rf CODH’s severely attenuated CO oxidation activity (13 mU/mg vs. 900 U/mg for the well-studied Ch CODH-II). EPR spectroscopy reveals unique oxidised C-cluster states not previously characterised in CODHs. Mirror tree analysis hints to co-evolution between clade B CODHs and associated ABC transporter substrate-binding proteins, suggesting these enzymes function in metabolism of substrates imported via the ABC transporter module. All findings indicate evolutionary repurposing of the CODH scaffold for alternative physiological functions.

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.

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 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 electron beam at room temperature. In this study, we developed a glove-box assisted cryo-transfer workflow for preparation of EM grids in 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 a feature of Na-valproate polyhedral chains. Our results provide a robust framework for 3D ED analysis of air-sensitive crystals, greatly enhancing its utility across various scientific disciplines.

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.

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.