Chemically Distinct Ni Sites in the A-Cluster in Subunit β of the Acetyl-CoA Decarbonylase/Synthase Complex from Methanosarcina thermophila:  Ni L-Edge Absorption and X-ray Magnetic Circular Dichroism Analyses

Abstract

The 5-subunit-containing acetyl-CoA decarbonylase/synthase (ACDS) complex plays an important role in methanogenic Archaea that convert acetate to methane, by catalyzing the central reaction of acetate C-C bond cleavage in which acetyl-CoA serves as the acetyl donor substrate reacting at the ACDS beta subunit active site. The properties of Ni in the active site A-cluster in the ACDS beta subunit from Methanosarcina thermophila were investigated. A recombinant, C-terminally truncated form of the beta subunit was employed, which mimics the native subunit previously isolated from the ACDS complex, and contains an A-cluster composed of an [Fe(4)S(4)] center bridged to a binuclear Ni-Ni site. The electronic structures of these two Ni were studied using L-edge absorption and X-ray magnetic circular dichroism (XMCD) spectroscopy. The L-edge absorption data provided evidence for two distinct Ni species in the as-isolated enzyme, one with low-spin Ni(II) and the other with high-spin Ni(II). XMCD spectroscopy confirmed that the species producing the high-spin signal was paramagnetic. Upon treatment with Ti(3+) citrate, an additional Ni species emerged, which was assigned to Ni(I). By contrast, CO treatment of the reduced enzyme converted nearly all of the Ni in the sample to low-spin Ni(II). The results implicate reaction of a high-spin tetrahedral Ni site with CO to form an enzyme-CO adduct transformed to a low-spin Ni(II) state. These findings are discussed in relation to the mechanism of C-C bond activation, in connection with the model of the beta subunit A-cluster developed from companion Ni and Fe K edge, XANES, and EXAFS studies.

Tight Coupling of Partial Reactions in the Acetyl-CoA Decarbonylase/Synthase (ACDS) Multienzyme Complex from Methanosarcina thermophila

“…The signal is considered to result from the formation of a Ni p 1ϩ -CO species and has been designated the "NiFeC" signal because of hyperfine broadening by 57 Fe, 61 Ni, and 13 CO. Under various conditions, two different and partially overlapping forms of the NiFeC signal have been detected (designated signal I and II) in which signal I has higher g values on average than signal II.…”

“…Reaction 1 is composed of several partial reactions (Scheme 1), which include acetyl group transfer, decarbonylation, methyl group transfer, and CO oxidation, catalyzed by different metalloprotein/enzyme subcomponents that can be released in active forms by partial proteolytic digestion of the complex (8 -10). Evidence indicates that the ␤ subunit contains an intact A cluster (11)(12)(13), which encompasses a binuclear Ni-Ni center bridged by a cysteine thiolate to an [Fe 4 S 4 ] cluster (14,15). The nickel atom proximal to the [Fe 4 S 4 ] cluster, designated Ni p , is coordinated by three -cysteine thiolates, one of which is shared with iron in the [Fe 4 S 4 ] cluster and with the other two in shared coordination with the distal Ni d .…”

Acetyl‐coenzyme A Synthases and Nickel‐Containing Carbon Monoxide Dehydrogenases

“…The A ox and A red-act states have been studied using Ni and Fe K-edge XAS, Ni L-edge XAS as well as soft X-ray MCD [62,63]. In the A ox state, 50% of spectral intensity was attributed to LS Ni 2ϩ and assigned to Ni d , while the other 50% was attributed to HS Ni 2ϩ and assigned to Ni p .…”

X-Treme beamline at SLS: X-ray magnetic circular and linear dichroism at high field and low temperature

“…Since its early times, XMCD has been widely used for the study of a variety of questions related to magnetism such as: magnetic anisotropy (Stö hr & Konig, 1995;Stö hr, 1999) and exchange bias at interfaces; element-specific magnetic properties of bimetallic paramagnetic molecules (Arrio et al, 1999); magnetic anisotropies, spin and orbital moments of single adatoms (Gambardella et al, 2003), of a submonolayer of singlemolecule magnets (Mannini et al, 2009) or organometallic molecules on magnetic surfaces (Wä ckerlin et al, 2010). XMCD has also been used in areas as wide as mineralogy (Pattrick et al, 2001), environmental sciences (Coker et al, 2006) and in biological systems such as metalloproteins (Funk et al, 2004(Funk et al, , 2005. A few characteristics that make this technique unique are its element specificity, the direct access to the states responsible for the chemical bonding and magnetic properties as well as the possibility to extract quantitative information on the spin and orbital moment separately through sum rules (Thole et al, 1992;Carra et al, 1993).…”