The Solvent-Exposed Fe-S D-Cluster Contributes to Oxygen-Resistance in Ni-Fe Carbon Monoxide Dehydrogenase.
Publication Type:Journal Article
Source:ACS Catal, Volume 10, Issue 13, p.7328-7335 (2020)
<p>Ni-Fe CO-dehydrogenases (CODHs) catalyze the conversion between CO and CO 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-sensitive and are always manipulated under strictly anaerobic conditions. However, the CODH from (Dv) appears unique: exposure to micromolar concentrations of O on the minutes-time scale 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-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 for increasing periods of time provide snapshots of [4Fe-4S] D-cluster degradation. We further show that the WT enzyme purified under conditions retains 30% activity relative to a fully 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-tolerance of this enzyme. Together, these results relate O-sensitivity with the details of the protein structure in this family of enzymes.</p>