Martin Högbom

Azole resistance in Candida species is often caused by the overexpression of Cdr1. Despite its clinical relevance, the structural basis for its ATP-driven efflux pump function remains elusive. We present four high-resolution cryo-EM structures of Candida glabrata Cdr1 obtained in the absence and presence of ATP-Mg²⁺, itraconazole, and vanadate, capturing distinct nucleotide- and substrate-occupancy states. Additional low-population cryo-EM conformers were unveiled by 3D variability analysis, offering a detailed view of structural transitions linked with ATP-hydrolysis. The motion shows a 4 Å piston-like retraction of the C-helix from the γ-phosphate/vanadate of the hydrolyzed ATP. This causes the nearby transmembrane helix-1 (TMH-1) to open the drug-binding site via lateral displacement and unwinding of the inner-leaflet region of TMH-2. A reverse ‘squeeze-and-push’ motion of TMH-2 possibly drives substrate extrusion. High-resolution structures also reveal how itraconazole adapts its shape to fit into the drug-binding site. Our findings provide a dynamic structural framework for Cdr1-mediated azole resistance and the conserved chemo-mechanical cycle of ABC proteins, including non-membranous members.

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.

Radical chemistry, once deemed too reactive for biological systems, is delicately controlled within metalloenzymes to catalyze challenging chemical transformations under physiological conditions. This review explores the diverse strategies employed by metalloenzymes to generate, stabilize, and utilize highly reactive radical intermediates. We discuss amino acid–based radicals (tyrosyl, tryptophan, cysteinyl, glycyl, and DOPA), radicals derived from molecular oxygen (in heme, manganese, copper, and nonheme iron enzymes), and cofactor-based radicals (specifically adenosylcobalamin and radical S -adenosylmethionine enzymes). We devote special attention to ribonucleotide reductases as a prime example of evolutionary convergence of radical mechanisms. The interplay among redox-active cofactors, metal ions, and protein scaffolds highlights nature's ingenuity in generating, controlling, and utilizing radicals. We also discuss emerging themes and open questions, emphasizing how advances in structural and spectroscopic techniques continue to deepen our understanding of these complex and vital enzymatic processes.

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.

Lipopolysaccharides are important components of the gram-negative bacterial cell envelope that are involved in immune evasion and act as a protective barrier. Employing cryo-electron microscopy, we resolved the structure and dynamics of FepE, the copolymerase component of the Wzy-dependent pathway, responsible for the length modulation of very long O-antigen molecules. Comparison of the interior volumes of related copolymerases’ periplasmic domains with the volume of hydrated sugars suggests that the size of the periplasmic domain controls the length of the O-antigen, implying that polysaccharide chain polymerization occurs inside the copolymerase periplasmic domain. Moreover, we show the opening of the FepE complex as well as other large mechanistically relevant movements. The opening of the complex presents an attractive corridor for the release of completed polysaccharide chains.

Cytochrome bd is a terminal oxidase expressed under low oxygen conditions and central for the survival of many pathogens. Here, we characterize the cyt bd -II from Mycobacterium smegmatis , a member of a hitherto uncharacterized evolutionary group (qOR-2) of bd oxidases, by combining biochemical studies with cryo-electron microscopy (cryo-EM), and multiscale simulations. Overexpressing the appCB operon in its native host led to production of a highly active bd -II ( k _obs = 30 e ⁻ s ⁻¹ ) that together with a high-resolution (2.8 Å) cryo-EM structure and multiscale simulations reveal unique proton pathways and oxygen channels responsible for its function. We propose that a pH-dependent molecular switch, involving coordination changes of heme d and surrounding bulky residues regulate substrate access into the active site. Taken together, our findings provide detailed mechanistic insight of qOR-2 type bd oxidases, and a basis for understanding the evolution of the superfamily.

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.