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.

Nickel in Subunit β of the Acetyl-CoA Decarbonylase/Synthase Multienzyme Complex in Methanogens

“…Multiple thiolate ligation to each nickel was indicated by the high values of molar absorptivity of the charge transfer bands formed upon nickel reconstitution. Characteristic UV-visible difference spectra obtained for binding of three different metals (Co 2ϩ , Ni 2ϩ , and Cu 2ϩ ) also implied multiple thiolate ligation, consistent with findings from extended x-ray absorption fine structure (EXAFS) studies on the related clostridial CODH/ ACS ␣ metallosubunit of nickel coordination by more than one sulfur ligand (21,22). The strong effect of pH on the rate of nickel incorporation was also consistent with deprotonation of more than one thiol group in the process of nickel binding, and site-directed mutagenesis ( Fig.…”

Evidence for Intersubunit Communication during Acetyl-CoA Cleavage by the Multienzyme CO Dehydrogenase/Acetyl-CoA Synthase Complex from Methanosarcina thermophila

“…The requirement for the ␣⑀ component in forming the NiFeC EPR signal also can be rationalized. Cluster A has a low midpoint potential of approximately Ϫ530 mV (44), and reduction of the catalytically active form of Cluster A appears to require CO (45). Therefore, as shown in Fig.…”

Genetic Construction of Truncated and Chimeric Metalloproteins Derived from the α Subunit of Acetyl-CoA Synthase from Clostridium thermoaceticum

“…The exact coordination environment of the Ni is not known, but it appears to consist of ~ 2 S and ~ 2 N donors (from cysteine and histidine residues), organized in a geometry that leaves two open cis coordination sites. 35,36 These sites are thought to bind CO and a methyl group during catalysis. 28,29 A hypothetical redox-active cystine/cysteine disulfide/dithiol called the D-site may facilitate methyl group transfer.…”