A unique [Ni-Fe-S] cluster (C-cluster) constitutes the active center of Ni-containing carbon monoxide dehydrogenases (CODHs). His(261), which coordinates one of the Fe atoms with Cys(295), is suggested to be the only residue required for Ni coordination in the C-cluster. To evaluate the role of Cys(295), we constructed CODH-II variants. Ala substitution for the Cys(295) substitution resulted in the decrease of Ni content and didn't result in major change of Fe content. In addition, the substitution had no effect on the ability to assemble a full complement of [Fe-S] clusters. This strongly suggests Cys(295) indirectly and His(261) together affect Ni-coordination in the C-cluster. (C) 2013 Elsevier Inc. All rights reserved..