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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

Ni-containing CO-dehydrogenases (CODHs) allow some microorganisms to couple ATP synthesis to CO oxidation, or to use either CO or CO 2 as a source of carbon. The recent detailed characterizations of some of them has evidenced a great diversity in terms of catalytic properties and resistance to O 2. In an effort to increase the number of available CODHs, we have heterologously produced in Desulfovibrio fructosovorans, purified and characterized the two CooS-type CODHs (CooS1 and CooS2) from the hyperthermophilic archaeon Thermococcus sp. AM4 (Tc). We have also crystallized CooS2, which is coupled in vivo to a hydrogenase. CooS1 and CooS2 are homodimers, and harbour five metalloclusters: two NiFe 4 S 4 C clusters, two [4Fe4S] B clusters and one interfacial [4Fe4S] D cluster. We show that both are dependent on a maturase, CooC1 or CooC2, which is interchangeable. The homologous protein CooC3 does not allow Ni insertion in either CooS. The two CODHs from Tc have similar properties: they can both oxidize and produce CO. The Michaelis constants (K m) are in the microM range for CO and in the mM range (CODH 1) or above (CODH 2) for CO 2. Product inhibition is observed only for CO 2 reduction, consistent with CO 2 binding being much weaker than CO binding. The two enzymes are rather O 2 sensitive (similarly to CODH II from Carboxydothermus hydrogenoformans), and react more slowly with O 2 than any other CODH for which these data are available.