The Solvent-Exposed Fe–S D-Cluster Contributes to Oxygen-Resistance in Desulfovibrio vulgaris Ni–Fe Carbon Monoxide Dehydrogenase

Elizabeth Wittenborn , Chloé Guendon (1) , Mériem Merrouch (1) , Martino Benvenuti (1) , Vincent Fourmond (1) , Christophe Léger (1) , Catherine Drennan , Sébastien Dementin (1)

1 BIP - Bioénergétique et Ingénierie des Protéines

Ni-Fe CO-dehydrogenases (CODHs) catalyze the conversion between CO and CO 2 using a chain of Fe-S clusters to mediate long-range electron transfer. One of these clusters, the D-cluster, is surface-exposed and serves to transfer electrons between CODH and external redox partners. These enzymes tend to be extremely O 2-sensitive and are always manipulated under strictly anaerobic conditions. However, the CODH from Desulfovibrio vulgaris (Dv) appears unique: exposure to micromolar concentrations of O 2 on the minutes-timescale only reversibly inhibits the enzyme, and full activity is recovered after reduction. Here we examine whether this unusual property of Dv CODH results from the nature of its D-cluster, which is a [2Fe-2S] cluster, instead of the [4Fe-4S] cluster observed in all other characterized CODHs. To this aim we produced and characterized a Dv CODH variant where the [2Fe-2S] D-cluster is replaced with a [4Fe-4S] D-cluster through mutagenesis of the D-cluster-binding sequence motif. We determined the crystal structure of this CODH variant to 1.83-Å resolution and confirmed the incorporation of a [4Fe-4S] D-cluster. We show that upon long-term O 2-exposure, the [4Fe-4S] D-cluster degrades whereas the [2Fe-2S] D-cluster remains intact. Crystal structures of the Dv CODH variant exposed to O 2 for increasing periods of time provide snapshots of [4Fe-4S] D-cluster degradation. We further show that the WT enzyme purified under aerobic conditions 1 Author accepted manuscript of doi:10.1021/acscatal.0c00934 retains 30% activity relative to a fully anaerobic purification, compared to 10% for the variant, and the WT enzyme loses activity more slowly than the variant upon prolonged aerobic storage. The D-cluster is therefore a key site of irreversible oxidative damage in Dv CODH, and the presence of a [2Fe-2S] D-cluster contributes to the O 2-tolerance of this enzyme. Together, these results relate O 2-sensitivity with the details of the protein structure in this family of enzymes.

Mots clés: CO-dehydrogenase; Iron-Sulfur cluster; Carbon monoxide; Oxygen-sensitivity; Oxidative damage; Crystallography; Protein film voltammetry

Domaines: Sciences du Vivant [q-bio]

Elizabeth Wittenborn, Chloé Guendon, Mériem Merrouch, Martino Benvenuti, Vincent Fourmond, et al.. The Solvent-Exposed Fe–S D-Cluster Contributes to Oxygen-Resistance in Desulfovibrio vulgaris Ni–Fe Carbon Monoxide Dehydrogenase. ACS Catalysis, 2020, 10 (13), pp.7328-7335. ⟨10.1021/acscatal.0c00934⟩. ⟨hal-02936282⟩