Nickel-dependent oligomerization of the alpha subunit of acetyl-coenzyme a synthase/carbon monoxide dehydrogenase

Abstract

After activation with NiCl2, the recombinant alpha subunit of the Ni-containing alpha2beta2 acetyl-CoA synthase/carbon monoxide dehydrogenase (ACS/CODH) catalyzes the synthesis of acetyl-CoA from CO, CoA, and a methyl group donated from the corrinoid-iron-sulfur protein (CoFeSP). The alpha subunit has two conformations (open and closed), and contains a novel [Fe4S4]-[Nip Nid] active site in which the proximal Nip ion is labile. Prior to Ni activation, recombinant apo-alpha contain only an Fe4S4 cluster. Ni-activated alpha subunits exhibit catalytic, spectroscopic and heterogeneity properties typical of alpha subunits contained in ACS/CODH. Evidence presented here indicates that apo-alpha is a monomer whereas Ni-treated alpha oligomerizes, forming dimers and higher molecular weight species including tetramers. No oligomerization occurred when apo-alpha was treated with Cu(II), Zn(II), or Co(II) ions, but oligomerization occurred when apo-alpha was treated with Pt(II) and Pd(II) ions. The dimer accepted only 0.5 methyl group/alpha and exhibited, upon treatment with CO and under reducing conditions, the NiFeC EPR signal quantifying to 0.4 spin/alpha. Dimers appear to consist of two types of alpha subunits, including one responsible for catalytic activity and one that provides a structural scaffold. Higher molecular weight species may be similarly constituted. It is concluded that Ni binding to the A-cluster induces a conformational change in the alpha subunit, possibly to the open conformation, that promotes oligomerization. These interrelated events demonstrate previously unrealized connections between (a) the conformation of the alpha subunit; (b) the metal which occupies the proximal/distal sites of the A-cluster; and (c) catalytic activity.

Figure 1

Gel filtration chromatography of the α subunit of Acetyl-CoA Synthase. A, Apo-α; B, Apo-α after incubation with NiCl₂. C, sample from B after concentration and re-passage through the column. Thick lines through the data (circles) are composite simulations consisting of the sum of three Gaussian lines (individually shown as thin lines). Best-fit linewidths were determined by fitting the data in C; these linewidths were then fixed while fitting the relative areas under the peaks in A and B. Resolution times were allowed to float slightly for each dataset. The relative percentile areas for the {monomer, dimer and tetramer} for the simulation in A, B and C were {88, 12, 0}, {15, 38, 47}, and {48, 49, 3}. Flow was 1 mL/min such that mL and min units can be interchanged.

Figure 2

Sedimentation velocity results of apo-α (black) and apo-α after Ni has been added (gray). Shown are integral distributions plots of the van Holde – Weischet analysis. While the apo-form appears to be primarily homogeneous with a sedimentation coefficient consistent with a monomer, the increased sedimentation coefficient distributions obtained upon addition of nickel indicate the presence of higher oligomeric forms.

Figure 3

Electron paramagnetic resonance of the dimeric form of α (A), apo-α (B), and the dimer-dissociated monomeric form of α (C). Samples were treated with 1 atm CO and 2 mM dithionite. EPR conditions: temperature, 10 K; microwave power, 20 mW; microwave frequency, 9.48 GHz; modulation amplitude, 11.8 G; sweep time, 328 s; time constant, 20 ms.

Figure 4

Stopped-flow of methyl transfer from methyl corrinoid-iron-sulfur protein (CH₃-CoFeSP, 10 μM in all reactions) to the recombinant α subunit. Upper panel: A, dimer (10 μM); B, dimer-dissociated monomers (10 μM), obtained as eluted FPLC fractions of the dimers in A, after overnight incubation in 100 μM NiCl₂; C, apo-α (10 μM); D, dimer-dissociated monomer (10 μM). Lower panel: E, F, G, H, and I, dimers with concentrations of 20, 10, 5, 2.5 and 1.5 μM, respectively. All protein concentrations are in terms of monomeric α subunits after mixing. Reactions were monitored at 390 nm, and the buffer was 50 mM TrisHCl, pH 8.0. Ti³⁺-citrate (1 mM) was present in all of these experiments.

Figure 5

Size-exclusion chromatography of metal-ion-treated α subunits (16mg/ml). A, NiCl₂; B, CuCl₂; C, ZnSO₄; D, CoCl₂; E, (NH₃)₂[PdCl₄]; F, K₂[PtCl₄].

Figure 6

Amino acid alignment of the N-terminal domain of the α subunit with a region of the β subunit. The α sequence begins at Asp005 and ends at Lys120. The β sequence begins at Asp552 and ends at Lys669.