Biology subjects

Fourmond, V.

Publications and source records attributed to Fourmond, V..

4 recordsLinked to original sources

bioRxiv2026

A conserved isoleucine gates the diffusion of small ligands to the active site of NiFe CO-dehydrogenase

CO dehydrogenases (CODH) are metalloenzymes that reversibly oxidize CO to CO2, at a buried NiFe4S4 active site. The substrates, CO and CO2, need therefore to be transported through the protein matrix to reach the active site. The most likely pathway for intra-protein diffusion is the hydrophobic channel identified in the crystal structures. We used site-directed mutagenesis in an extensive manner to study the role of the highly conserved isoleucine 563 of Thermococcus sp. AM4 CODH2. Certain substitutions significantly change the biochemical properties of the enzyme (KM for CO, catalytic efficiency, product inhibition constant, catalytic bias...), and increase its resistance to the inhibitor O2, showing that isoleucine 563 plays a key role in determining access to the active site. The mutations have the same effects on the rates of binding of CO and O2 showing that the two molecules follow the same pathway and are not discriminated by the protein matrix. The I563F mutation decreases the bimolecular rate constant of inhibition by O2 15-fold, and increases the IC50 20-fold, which is the strongest improvement in O2 resistance reported so far.

biochemistry↗

bioRxiv2026

Correspondence on "Fortification of FeS Clusters Reshapes Anaerobic CO Dehydrogenase into an Air-Viable Enzyme ThroughMultilayered Sealing of O2 Tunnels"

In their recent communication in Angewandte Chemie (10.1002/anie.202508565), Suk Min Kim and coworkers have described the effect of modifying the gas channels of the CO dehydrogenase II from Carboxydothermus hydrogenoformans, an enzyme that oxidizes reversibly CO into CO2. Their goal was to use mutagenesis to slow down the arrival of O2 at the active site. They reported a large increase in the resistance against oxygen, one of the major barriers to the application of this extremely fast and efficient enzyme in biotechnological devices, with an increase in the IC50 of more than two orders of magnitudes for some variants, with only a minor impact on the affinity of the enzyme for CO. We have produced the same variants, and characterized them in depth using Protein Film Electrochemistry. We used an approach that has proven very useful to learn and understand about the reactivity of CO dehydrogenases (and other redox enzymes like hydrogenases) with O2. We found that, contrary to the claims by Kim and coworkers, the A559W and the A559W/V610H mutants are not more resistant than the WT against oxygen.

biochemistry↗

bioRxiv2023

Catalytic bias and redox-driven inactivation of ancestral FeFe hydrogenases from group B

The biodiversity of hydrogenases, the enzymes that oxidize and produce H2, is only just beginning to be explored. Here we use direct electrochemistry to characterize two enzymes from a subgroup of ancestral FeFe hydrogenases, defined by the presence of three adjacent cysteine residues near the active site: the third FeFe hydrogenase from Clostridium pasteurianum (CpIII) and the second from Megasphaera elsdenii (MeII). To examine the functional role of the unusual TSCCCP motif, which defines the group B2 and is replaced with TSCCP in group A hydrogenases, we also produced a CpIII variant where the supernumerary cysteine is deleted. CpIII and MeII inactivate under oxidative conditions in a manner that is distinct from all other previously characterized hydrogenases from group A. Our results suggest that the supernumerary cysteine allows the previously observed sulfide-independent formation of the Hinact state in these enzymes. We also evidence a second reversible, oxidative inactivation process. Because of their inactivation under oxidative conditions, these enzymes are inefficient H2 oxidation catalysts, but their active site itself is not tuned to make them more active in one particular direction.

biochemistry↗

bioRxiv2019

How thermophilic Gram-positive organisms perform extracellular electron transfer: characterization of the cell surface terminal reductase OcwA

Extracellular electron transfer is the key process underpinning the development of bioelectrochemical systems for the production of energy or added-value compounds. Thermincola potens JR is a promising Gram-positive bacterium to be used in these systems because it is thermophilic. In this paper we describe the structural and functional properties of the nonaheme cytochrome OcwA, which is the terminal reductase of this organism. The structure of OcwA, determined at 2.2[A] resolution shows that the overall-fold and organization of the hemes are not related to other metal reductases and instead are similar to that of multiheme cytochromes involved in the biogeochemical cycles of nitrogen and sulfur. We show that, in addition to solid electron acceptors, OcwA can also reduce soluble electron shuttles and oxyanions. These data reveal that OcwA can take the role of a respiratory swiss-army knife allowing this organism to grow in environments with rapidly changing availability of terminal electron acceptors without the need for transcriptional regulation and protein synthesis.\n\nImportanceThermophilic Gram-positive organisms were recently shown to be a promising class of organisms to be used in bioelectrochemical systems for the production of electrical energy. These organisms present a thick peptidoglycan layer that was thought to preclude them to perform extracellular electron transfer (i.e. exchange catabolic electrons with solid electron acceptors outside of the cell). In this manuscript we describe the structure and functional mechanisms of the multiheme cytochrome OcwA, the terminal reductase of the Gram-positive bacterium Thermincola potens JR found at the cell surface of this organism. The results presented here show that this protein is unrelated with terminal reductases found at the cell surface of other electroactive organisms. Instead, OcwA is similar to terminal reductases of soluble electron acceptors. Our data reveals that terminal oxidoreductases of soluble and insoluble substrates are evolutionarily related, providing novel insights into the evolutionary pathway of multiheme cytochromes.

biochemistry↗