Mo/W formate dehydrogenases catalyze the reversible reduction of CO 2 species to formate. It is thought that the substrate is CO 2 and not a hydrated species like HCO 3 À , but there is still no indisputable evidence for this, in spite of the extreme importance of the nature of the substrate for mechanistic studies. We devised a simple electrochemical method to definitively demonstrate that the substrate of formate dehydrogenases is indeed CO 2 .

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.

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