Ligand binding at the A-cluster in full-length or truncated acetyl-CoA synthase studied by X-ray absorption spectroscopy

Abstract

Bacteria integrate CO2 reduction and acetyl coenzyme-A (CoA) synthesis in the Wood-Ljungdal pathway. The acetyl-CoA synthase (ACS) active site is a [4Fe4S]-[NiNi] complex (A-cluster). The dinickel site structure (with proximal, p, and distal, d, ions) was studied by X-ray absorption spectroscopy in ACS variants comprising all three protein domains or only the C-terminal domain with the A-cluster. Both variants showed two square-planar Ni(II) sites and an OH- bound at Ni(II)p in oxidized enzyme and a H2O at Ni(I)p in reduced enzyme; a Ni(I)p-CO species was induced by CO incubation and a Ni(II)-CH3- species with an additional water ligand by a methyl group donor. These findings render a direct effect of the N-terminal and middle domains on the A-cluster structure unlikely.

Characterization of Methyl- and Acetyl-Ni Intermediates in Acetyl CoA Synthase Formed during Anaerobic CO2 and CO Fixation

“…A prominent backscattering peak owing to the Ni p –Ni d and Ni p –Fe contributions is also present in the second shell of the Fourier-transform Ni EXAFS, which was also fit to 2.98 Å, confirming the proper metal loading at the active site. Additionally, sharpening of the Ni K-rising-edge feature on conversion of A Ox to A Me is consistent with increasing planarity at the Ni sites suggested by theoretical calculations (see below) that show that the planarity of Ni d remains unaltered while Ni p approaches a square-planar configuration upon methyl binding. − It is important to note here that our XAS results contrast with prior XAS studies of the A Me form of Carboxydothermus hydrogenoformans ACS, which showed a rising-edge shifted to lower-energy relative to A Ox , and assignment of a weak ∼2.4 Å Ni–Me, bound in the apical position of a square pyramid. The study lacks from supporting kinetic or spectroscopic quantification of the percentage methylation as well as the extent of metal loading at the Ni p , Ni d , and [4Fe–4S] 2+ cluster sites and comparison with the data presented here suggest only modest methylation in the experiments on the C.…”

Theoretical Studies of Acetyl-CoA Synthase Catalytic Mechanism

“…The data in Table 6 for protonation on the hydroxyl ligand in A ox and A red1 shows that pK a values are relatively low for the oxidized form and high for one-electron reduced form. This is in accordance with the experimental findings that the oxidized form has a hydroxyl ligand and one-electron reduced a water one [56]. The pK a values for A red2 with ligands are quite large and significantly lower for the dielectric constant 80.…”

“…At the same time, it can be found that the Ni p -Fe1 distance is very small (2.243 Å) in the PDB ID: 6YTT crystal structure [46] where there is no ligand on Ni p and in turn very long in the crystal structure PDB ID: 6X5K [36] with the carbonyl ligand. This is not the case in the EXAFS results for the CO ligand, where this distance is equal to 2.80 Å [56] (Table 2). If we look at the Ni p -Fe1 distances obtained from the calculations, it can be seen that the M1 model with CO gives values similar to the crystal structure and the M2 model to EXAFS [56]; however, both calculated values are longer than in the experimental case.…”

“…This is not the case in the EXAFS results for the CO ligand, where this distance is equal to 2.80 Å [56] (Table 2). If we look at the Ni p -Fe1 distances obtained from the calculations, it can be seen that the M1 model with CO gives values similar to the crystal structure and the M2 model to EXAFS [56]; however, both calculated values are longer than in the experimental case. The carbonyl bound Ni(I) ion has a tetrahedral coordination sphere which can be seen from the valence angles around Ni p (Table 2).…”

“…Several forms of the A-cluster relevant to the catalytic reaction were characterized structurally or spectroscopically [8,36,37,46,[56][57][58][59][60][61][62][63][64][65]. They can be described by the oxidation state, electronic configuration, spin, and ligands attached to Ni p , as shown in Table 1.…”