The A-Cluster in Subunit β of the Acetyl-CoA Decarbonylase/Synthase Complex from <i>Methanosarcina thermophila</i>: Ni and Fe K-Edge XANES and EXAFS Analyses

The acetyl-CoA decarbonylase/synthase (ACDS) complex catalyzes the cleavage of acetyl-CoA in methanogens that metabolize acetate to CO<sub>2</sub> and CH<sub>4</sub>, and also carries out acetyl-CoA synthesis during growth on one-carbon substrates. The ACDS complex contains five subunits, among which β possesses an Ni−Fe−S active-site metal cluster, the A-cluster, at which reaction with acetyl-CoA takes place, generating an acetyl-enzyme species poised for C−C bond cleavage. We have used Ni and Fe K fluorescence XANES and EXAFS analyses to characterize these metals in the ACDS β subunit, expressed as a C-terminally shortened form. Fe XANES and EXAFS confirmed the presence of an [Fe<sub>4</sub>S<sub>4</sub>] cluster, with typical Fe−S and Fe−Fe distances of 2.3 and 2.7 Å respectively. An Fe:Ni ratio of ∼2:1 was found by Kαβ fluorescence analysis, indicating 2 Ni per [Fe<sub>4</sub>S<sub>4</sub>]. Ni XANES simulations were consistent with two distinct Ni sites in cluster A, and the observed spectrum could be modeled as the sum of separate square planar and tetrahedral Ni sites. Treatment of the β subunit with Ti<sup>3+</sup> citrate resulted in shifts to lower energy, implying significant reduction of the [Fe<sub>4</sub>S<sub>4</sub>] center, along with conversion of a smaller fraction of Ni(II) to Ni(I). Reaction with CO in the presence of Ti<sup>3+</sup> citrate generated a unique Ni XANES spectrum, while effects on the Fe-edge were not very different from the reaction with Ti<sup>3+</sup> alone. Ni EXAFS revealed an average Ni coordination of 2.5 S at 2.19 Å and 1.5 N/O at 1.89 Å. A distinct feature at ∼2.95 Å most likely results from Ni−Ni interaction. The methanogen β subunit A-cluster is proposed to consist of an [Fe<sub>4</sub>S<sub>4</sub>] cluster bridged to an Ni−Ni center with one Ni in square planar geometry coordinated by 2 S + 2 N and the other approximately tetrahedral with 3 S + 1 N/O ligands. The electronic consequences of two distinct Ni geometries are discussed.