Spectroscopic, Redox, and Structural Characterization of the Ni-Labile and Nonlabile Forms of the Acetyl-CoA Synthase Active Site of Carbon Monoxide Dehydrogenase

Abstract

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 study was to elucidate the redox and spectroscopic properties of these A-cluster forms and thereby understand their structural and functional differences. The Ni-labile form 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°‘ for the Ni2+/Ni1+ couple, thereby rendering CO and triquat effective reductants. Dithionite did not reduce the Ni-labile form, though its addition to CO/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 catalysis. It may also transfer electrons to and from the redox-active D site during reductive activation.