The [NiFe]-CODH from Rhodospirillum rubrum contains [4Fe4S] clusters that allow electron transfer from the buried active sites to the protein surface. Among them, the role of the D-cluster, located at the dimer interface is still not fully understood. In this study, the removal of the D-cluster by site-directed mutagenesis revealed remarkable features in the behavior of the enzyme. Quantitative analysis and spectroscopic studies unveiled the suppression of D-cluster in the mutants and the influence on other metal cofactors. Furthermore, the CO oxidation activity in solution measured in the presence of methylviologen is almost completely abolished in the mutants. Conversely, direct electrochemistry at a functionalized carbon nanotube electrode shows that the mutants are still catalytically active reaching reduced but significant current densities of 0.7 mA cm