Spectroscopic, redox, and structural characterization of the Ni-labile and nonlabile forms of the acetyl-CoA synthase active site of carbon monoxide dehydrogenase

The α subunit of carbon monoxide dehydrogenase from: Clostridium thermoaceticum Was isolated, treated as described below, and examined by XAS, EPR, and UV-vis spectroscopies. This subunit contains the active site for acetyl-coenzyme A synthesis, the A-cluster, a Ni ion bridged to an Fe₄S₄ cube. Populations of α subunits contain two major forms of A-clusters, a catalytically active form called Ni-labile and an inactive form called nonlabile. The objective of this stud was to elucidate the redox and spectroscopic properties of could be reduced either by CO and a catalytic amount of native enzyme or by electrochemically reduced triquat in the presence of CO. The Ni²⁺ component of the Ni-labile form reduced to Ni¹⁺ and bound CO. CO-binding raised E(o') for the Ni²⁺/Ni¹⁺ couple, thereby rendering CO and triquat effective reductants. Dithionite did not reduce the Ni-labile form, though its addition to CD/CODH-reduced Ni-labile clusters caused an intracluster electron transfer from the Ni¹⁺ to the [Fe₄S₄]²⁺ cluster. Dithionite reduced the [Fe₄S₄]²⁺ component of the nonlabile form, as well as the cluster of the Ni-labile form once Ni was removed. Ni may not be bridged to the cube in the nonlabile form. XAS reveals that the Ni in the nonlabile form has a distorted square-planar geometry with two N/O scatters at 1.87 Å and two S scatters at 2:20 Å. The [Fe₄S₄]²⁺ portion of Ni-labile A-clusters may maintain the Ni in a geometry conducive to reduction, CO and methyl group binding, and the migratory-insertion step used in catalysts. It may also transfer electrons to and from the redox-active D site during reductive activation.