A conserved isoleucine gates the diffusion of CO and O 2 to the active site of NiFe COdehydrogenase
Abstract
CO dehydrogenases (CODH) are metalloenzymes that reversibly oxidize CO to CO 2 , at a buried NiFe 4 S 4 active site. The substrates, CO and CO 2 , 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 (K M for CO, catalytic efficiency, product inhibition constant, catalytic bias…), and increase its resistance to the inhibitor O 2 , 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 O 2 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 O 2 15-fold, and increases the IC50 20-fold. This is the strongest improvement in O 2 resistance reported so far, but it comes at the cost of reduced substrate affinity.
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BibTeX
@article{opdam2026conserved,
title = {{A conserved isoleucine gates the diffusion of CO and O 2 to the active site of NiFe COdehydrogenase}},
author = {Laura Opdam and Marta Meneghello and Chloé Guendon and Jade Chargelègue and Andrea Fasano and Aurore Jacq-Bailly and Christophe Léger and Vincent Fourmond},
journal = {Angewandte Chemie International Edition},
year = {2026},
month = sep,
doi = {10.1002/anie.6262238},
url = {https://hal.science/hal-05747725v1},
note = {CC BY 4.0},
}
RIS
TY - JOUR
TI - A conserved isoleucine gates the diffusion of CO and O 2 to the active site of NiFe COdehydrogenase
AU - Laura Opdam
AU - Marta Meneghello
AU - Chloé Guendon
AU - Jade Chargelègue
AU - Andrea Fasano
AU - Aurore Jacq-Bailly
AU - Christophe Léger
AU - Vincent Fourmond
PY - 2026
DA - 2026/09/11
JO - Angewandte Chemie International Edition
DO - 10.1002/anie.6262238
UR - https://hal.science/hal-05747725v1
AB - CO dehydrogenases (CODH) are metalloenzymes that reversibly oxidize CO to CO 2 , at a buried NiFe 4 S 4 active site. The substrates, CO and CO 2 , 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 (K M for CO, catalytic efficiency, product inhibition constant, catalytic bias…), and increase its resistance to the inhibitor O 2 , 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 O 2 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 O 2 15-fold, and increases the IC50 20-fold. This is the strongest improvement in O 2 resistance reported so far, but it comes at the cost of reduced substrate affinity.
SN - 1433-7851
SN - 1521-3773
ER -