Maximilian Böhm

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.

Carbon monoxide dehydrogenases (CODHs) are metalloenzymes central to microbial CO metabolism and CO ₂ fixation. We report the heterologous production and characterisation of Clostridium pasteurianum BC1 CODH‐III ( Cp ^BC1 CODH‐III), from the phylogenetic clade E, co‐expressed with its maturation machinery CooCTJ. Cp ^BC1 CODH‐III shows moderate CO oxidation (150 U/mg) and CO ₂ reduction (0.568 U/mg) activities. Electron paramagnetic resonance (EPR) spectroscopy under varying redox conditions identified a rhombic signal (g _av ≈ 1.97; g ₁ ≈ 2.06, g ₂ ≈ 1.95, g ₃ ≈ 1.91), characteristic of reduced B‐clusters, and C‐clusters at different redox levels (g ₃ ≈ 1.76, g ₃ ≈ 1.73), indicative of bound CO ₂ and OH. Investigation of maturation effects showed that co‐expression of CooCTJ stabilised Cp ^BC1 CODH‐III production, but did not enhance maximum activity, which was primarily influenced by nickel availability. Comparative operon analysis with the well‐studied clade F Rhodospirillum rubrum CODH ( Rr CODH) revealed high structural similarity in CODH and CooC, but significant divergence in CooJ, with conserved metal‐binding regions identified via AlphaFold3 modelling and dot plot analysis. Cp ^BC1 CODH‐III represents a unique example of a clade E CODH within a clade F genomic context, demonstrating intrinsic robustness in maturation and activity.

Carbon monoxide dehydrogenases containing nickel-iron active sites ([NiFe]-CODHs) catalyze the reversible oxidation of CO to CO ₂ , representing key targets for biocatalytic CO ₂ reduction. Despite dramatic differences in catalytic rates and O ₂ tolerance between CODH variants, the molecular basis for this functional diversity remains poorly understood. We applied comparative genomics and synteny analysis to investigate the biochemical roles of CODH clades A-F using 1376 CODH and 1545 hybrid cluster protein sequences. Around 30% of genomes encode multiple CODH isoforms. Analysis revealed distinct gene clustering patterns correlating with biochemical function. Clades A, E, and F exhibit a degree of distributional exclusivity. Clades C and D frequently co-occur with active CODHs, suggesting auxiliary roles. Operon architecture analysis revealed functional specialization: clade A links to acetyl-CoA synthase; clades A, E, and F contain essential maturation machinery (CooC, CooJ, CooT) correlating with catalytic activity; clade B associates with transporters; clade C with electron transfer partners; clade D with transcriptional regulators. High CODH-HCP co-occurrence (except clade A) suggests functional or environmental interdependency. These findings establish clades A, E, and F as primary biocatalyst targets while defining regulatory functions for clades C and D, providing a genomics framework for predicting CODH phenotypes.

Carbon monoxide dehydrogenases (CODHs) are metalloenzymes central to microbial CO metabolism and CO ₂ fixation. We report the heterologous production and characterisation of Clostridium pasteurianum BC1 CODH-III ( Cp ^BC1 CODH-III), from the phylogenetic clade E, co-expressed with its maturation machinery CooCTJ. Cp ^BC1 CODH-III shows moderate CO oxidation (150 U/mg) and CO ₂ reduction (0.568 U/mg) activities. Electron paramagnetic resonance (EPR) spectroscopy under varying redox conditions identified a rhombic signal at g ≈ 2.0, characteristic of reduced B-clusters, and a C-clusters at different stages (g ≈ 1.75, g ≈ 1.72), indicative of a bound CO ₂ . Investigation of maturation effects showed that co-expression of CooCTJ stabilised Cp ^BC1 CODH-III production, but did not enhance maximum activity, which was primarily influenced by nickel availability. Comparative operon analysis with the well-studied clade F Rhodospirillum rubrum CODH ( Rr CODH) revealed high structural similarity in CODH and CooC, but significant divergence in CooJ, with conserved metal-binding regions identified via AlphaFold3 modelling and dot plot analysis. Cp ^BC1 CODH-III represents a unique example of a clade E CODH within a clade F genomic context, demonstrating intrinsic robustness in maturation and activity

Carbon monoxide dehydrogenases containing nickel-iron active sites ([NiFe]-CODHs) catalyze the reversible oxidation of CO to CO2, representing key targets for biocatalytic CO2 reduction. Despite dramatic differences in catalytic rates and O2 tolerance between CODH variants, the molecular basis for this functional diversity remains poorly understood. We applied comparative genomics and synteny analysis to investigate the biochemical roles of CODH clades A-F using 1,376 CODH and 1,545 hybrid cluster protein sequences. Around 30% of genomes encode multiple CODH isoforms. Analysis revealed distinct gene clustering patterns correlating with biochemical function. Clades A, E, and F exhibit a degree of distributional exclusivity. Clades C and D frequently co-occur with active CODHs, suggesting auxiliary roles. Operon architecture analysis revealed functional specialization: clade A links to acetyl-CoA synthase; clades A, E and F contain essential maturation machinery (CooC, CooJ, CooT) correlating with catalytic activity; clade B associates with transporters; clade C with electron transfer partners; clade D with transcriptional regulators. High CODH-HCP co-occurrence (except clade A) suggests functional or environmental interdependency. These findings establish clades A, E and F as primary biocatalyst targets while defining regulatory functions for clades C and D, providing a genomics framework for predicting CODH phenotypes.

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.

Carbon monoxide dehydrogenases containing nickel-iron active sites ([NiFe]-CODHs) catalyze the reversible oxidation of CO to CO ₂ , representing key targets for biocatalytic CO ₂ reduction. Despite dramatic differences in catalytic rates and O ₂ tolerance between CODH variants, the molecular basis for this functional diversity remains poorly understood. We applied comparative genomics and synteny analysis to investigate the biochemical roles of CODH clades A-F using 1,376 CODH and 1,545 hybrid cluster protein sequences. Around 30% of genomes encode multiple CODH isoforms. Analysis revealed distinct gene clustering patterns correlating with biochemical function. Clades A, E, and F exhibit a degree of distributional exclusivity. Clades C and D frequently co-occur with active CODHs, suggesting auxiliary roles. Operon architecture analysis revealed functional specialization: clade A links to acetyl-CoA synthase; clades A, E and F contain essential maturation machinery (CooC, CooJ, CooT) correlating with catalytic activity; clade B associates with transporters; clade C with electron transfer partners; clade D with transcriptional regulators. High CODH-HCP co-occurrence (except clade A) suggests functional or environmental interdependency. These findings establish clades A, E and F as primary biocatalyst targets while defining regulatory functions for clades C and D, providing a genomics framework for predicting CODH phenotypes.

Photobiocatalytic CO2 reduction represents an attractive approach for conversion of solar light and abundant resources to value-added chemicals. However, the design of suitable systems requires a detailed understanding of the interaction between artificial photosensitizer and biocatalyst interface. In this work, we investigate the effect of surfactant charge utilized in the preparation of a phenoxazine-based organic molecule nanorods photosensitizer on the interaction with the carbon monoxide dehydrogenase II from Carboxydothermus hydrogenoformans within biohybrid assemblies for photobiocatalytic CO2 reduction into CO. Electrophoretic mobility shift assay in pair with cryogenic electron microscopy (Cryo-EM) and detailed physicochemical characterization are conducted to understand the interaction at the biohybrid interface in order to suggest the strategy for future functionalization of nanoparticles that fulfill the needs from biocatalyst for green fuel production.

Many known chemotherapeutic anticancer agents exhibit neutropenia as a dose-limiting side effect. In this paper we suggest a prodrug concept solving this problem for camptothecin (HO-cpt). The prodrug is programmed according to Boolean “AND” logic. It requires for its activation the simultaneous presence of triggers T1 (H2O2) and T2 (pH 8 in mitochondria: Mit). The level of H2O2 in cancer cells is higher than that in normal cells. Thus, T1 discriminates cancer cells from the majority of normal cells excluding neutrophils, known to produce elevated levels of H2O2. T2 discriminates cancer cells from neutrophils, since the former cells have a higher number of Mit. We demonstrated that our prodrug exhibits antitumor activity both in vitro and in vivo, but is not toxic to normal cell and neutrophils in contrast to known single trigger prodrugs and the parent drug HO-cpt.