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

Abstract

The acetyl-CoA decarbonylase/synthase (ACDS) complex catalyzes the cleavage of acetyl-CoA in methanogens that metabolize acetate to CO(2) and CH(4), and also carries out acetyl-CoA synthesis during growth on one-carbon substrates. The ACDS complex contains five subunits, among which beta 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 beta subunit, expressed as a C-terminally shortened form. Fe XANES and EXAFS confirmed the presence of an [Fe(4)S(4)] cluster, with typical Fe-S and Fe-Fe distances of 2.3 and 2.7 A respectively. An Fe:Ni ratio of approximately 2:1 was found by Kalphabeta fluorescence analysis, indicating 2 Ni per [Fe(4)S(4)]. 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 beta subunit with Ti(3+) citrate resulted in shifts to lower energy, implying significant reduction of the [Fe(4)S(4)] center, along with conversion of a smaller fraction of Ni(II) to Ni(I). Reaction with CO in the presence of Ti(3+) citrate generated a unique Ni XANES spectrum, while effects on the Fe-edge were not very different from the reaction with Ti(3+) alone. Ni EXAFS revealed an average Ni coordination of 2.5 S at 2.19 A and 1.5 N/O at 1.89 A. A distinct feature at approximately 2.95 A most likely results from Ni-Ni interaction. The methanogen beta subunit A-cluster is proposed to consist of an [Fe(4)S(4)] 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.

CO in methanogenesis

“…Although a structure is not available, a variety of spectroscopic studies suggest the A cluster is comprised of an Fe 4 S 4 center bridged to a binuclear Ni-Ni site (Gu et al 2003;Funk et al 2004) similar in structure (Fig. 8) to that proposed for the homolog from M. thermoacetica (Ragsdale 2007).…”

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

“…The reaction mixtures (360 l) were set up at 25°C and contained 100 M acetyl-CoA, 100 M 3Ј-dephospho-CoA, 100 mM KCl, 50 mM MOPS buffer, pH 6.7, and 50 M aquacobalamin prereduced with 136 M Ti 3ϩ NTA. All components except the CoA substrates and enzyme were assembled and incubated for 30 min to allow for reduction of B 12 . Thereafter, 3Ј-dephospho-CoA was added, followed by the enzyme, and the reaction was initiated by addition of acetyl-CoA.…”

“…In the reverse direction, cleavage of the acetyl C-C bond yields separate methyl and CO fragments bound to the A cluster. To measure the reversible fragmentation and reassembly of the A cluster acetyl species catalyzed by the M. thermophila ACDS complex, carbonyl exchange assays were performed using [1-14 C]acetyl-CoA as substrate in the presence of either 12 CO or 12 CO 2 . As shown in Fig.…”

“…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 .…”

Application of Stopped-Flow and Time-Resolved X-Ray Absorption Spectroscopy to the Study of Metalloproteins Molecular Mechanisms

“…Since XAS accurately reports the structure of metal-protein centers, early workers focused on providing additional high resolution structural and electronic information on crystallographically characterized samples. For instance, XAS enabled detailed structural investigation of metal active sites in imidazolonepropionase 25 , cytochrome P450 26,27 , CO dehydrogenase/acetyl-CoA synthase [28][29][30] , manganese catalases 31,32 , and lipoxygenase 33 by providing key insights into their electronic states and atomic structures. Moreover, insights into the enzymatic reaction mechanisms could be derived from XAS analysis, as was shown for tyrosine hydroxylase 34 , molybdenum(Mo)-nitogenase [35][36][37] , and farnesyltransferase 38 .…”