ja036602a_si_001.pdf (37.48 kB)
The A-Cluster in Subunit β of the Acetyl-CoA Decarbonylase/Synthase Complex from Methanosarcina thermophila: Ni and Fe K-Edge XANES and EXAFS Analyses
journal contribution
posted on 2003-12-17, 00:00 authored by Weiwei Gu, Simonida Gencic, Stephen P. Cramer, David A. GrahameThe acetyl-CoA decarbonylase/synthase (ACDS) complex catalyzes the cleavage of acetyl-CoA
in methanogens that metabolize acetate to CO2 and CH4, 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 [Fe4S4] 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 [Fe4S4]. 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 Ti3+ citrate resulted in shifts to lower energy, implying
significant reduction of the [Fe4S4] center, along with conversion of a smaller fraction of Ni(II) to Ni(I). Reaction
with CO in the presence of Ti3+ citrate generated a unique Ni XANES spectrum, while effects on the Fe-edge were not very different from the reaction with Ti3+ 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 [Fe4S4] 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.