SPECTROSCOPIC, REDOX, AND STRUCTURAL CHARACTERIZATION OF THE NI-LABILE AND NONLABILE FORMS OF THE ACETYL-COA SYNTHASE ACTIVE - SITE OF CARBON-MONOXIDE DEHYDROGENASE

The alpha subunit of carbon monoxide dehydrogenase from Clostridium th ermoaceticum 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 a subunits contain two major forms o f A-clusters, a catalytically active form called Ni-labile and an inac tive form called nonlabile. The objective of this study was to elucida te the redox and spectroscopic properties of these A-cluster forms and thereby understand their structural and functional differences. The N i-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 NiI+ and bo und CO. GO-binding raised E-o' for the Ni2+/Ni1+ couple, thereby rende ring CO and triquat effective reductants. Dithionite did not reduce th e Ni-labile form, though its addition to CO/CODH-reduced Ni-labile clu sters caused an intracluster electron transfer from the Ni1+ to the [F e4S4](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-p lanar geometry with two N/O scatters at 1.87 Angstrom and two S scatte rs at 2.20 Angstrom. The [Fe4S4](2+) portion of Ni-labile A-clusters m ay 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 durin g reductive activation.