Infrared and EPR spectroscopic characterization of a Ni(I) species formed by photolysis of a catalytically competent Ni(I)-CO intermediate in the acetyl-CoA synthase reaction

Abstract

Acetyl-CoA synthase (ACS) catalyzes the synthesis of acetyl-CoA from CO, coenzyme A (CoA), and a methyl group from the CH(3)-Co(3+) site in the corrinoid iron-sulfur protein (CFeSP). These are the key steps in the Wood-Ljungdahl pathway of anaerobic CO and CO(2) fixation. The active site of ACS is the A-cluster, which is an unusual nickel-iron-sulfur cluster. There is significant evidence for the catalytic intermediacy of a CO-bound paramagnetic Ni species, with an electronic configuration of [Fe(4)S(4)](2+)-(Ni(p)(+)-CO)-(Ni(d)(2+)), where Ni(p) and Ni(d) represent the Ni centers in the A-cluster that are proximal and distal to the [Fe(4)S(4)](2+) cluster, respectively. This well-characterized Ni(p)(+)-CO intermediate is often called the NiFeC species. Photolysis of the Ni(p)(+)-CO state generates a novel Ni(p)(+) species (A(red)*) with a rhombic electron paramagnetic resonance spectrum (g values of 2.56, 2.10, and 2.01) and an extremely low (1 kJ/mol) barrier for recombination with CO. We suggest that the photolytically generated A(red)* species is (or is similar to) the Ni(p)(+) species that binds CO (to form the Ni(p)(+)-CO species) and the methyl group (to form Ni(p)-CH(3)) in the ACS catalytic mechanism. The results provide support for a binding site (an "alcove") for CO near Ni(p), indicated by X-ray crystallographic studies of the Xe-incubated enzyme. We propose that, during catalysis, a resting Ni(p)(2+) state predominates over the active Ni(p)(+) species (A(red)*) that is trapped by the coupling of a one-electron transfer step to the binding of CO, which pulls the equilibrium toward Ni(p)(+)-CO formation.

Figure 1

Structure of the A-Cluster of ACS and the gas-binding site. (A) The structure is based on that of PDB ID1OAO (6), (B) The structure is based on molecule P of PDB ID 2Z8Y (7), which was generated from a crystal structure obtained at high pressures of Xe. In this structure, Cu was present in the proximal site (Cu_p) and Ni was present in the distal site Ni_d. Some of the hydrophobic residues are shown that were proposed (7) to form a gas-binding site (the “alcove”) near the binuclear center (Ni_p and Ni_d).

Figure 2

Paramagnetic mechanism of ACS. In this so-called “paramagnetic mechanism”, which is written simplistically as an ordered reaction (although it is a random order of CO or methyl addition), CO traps the thermodynamically unfavorable Ni¹⁺ state (A_red*). However, this state can be trapped at low temperatures by photolysis of the CO ligand. The A_red-CO state is favored because of CO backbonding to a low valent Ni and because ACS has a CO binding site (“alcove”) near Ni_p. Transmethylation generates an unstable methyl-Ni³⁺ state that accepts an electron to generate the stable methyl-Ni²⁺, which undergoes carbonyl insertion to generate an acetyl-Ni²⁺. As the acetyl group undergoes thiolysis by the nucleophilic attack of CoA, two electrons bifurcate: one goes into the internal electron transfer pathway and the other is used to regenerate Ni¹⁺. In the diamagnetic mechanism, so-called because none of the proposed intermediates are paramagnetic, the substrate(s) bind to a diamagnetic “Ni(0)” state to generate a diamagnetic methyl-Ni(II) or Ni(II)-CO species. The intramolecular electron transfer step would not be required in the “diamagnetic mechanism”. See the text for details.

Figure 3

Time dependence of IR absorbance changes upon photolysis of A_red-CO at 4.5 K. Inset: Development of features in the 2050–2150 cm⁻¹ region, using ¹²CO and ¹³CO. See the Materials and Methods section for details of the FTIR experiment. Time points for ¹²CO data are: 0 (black), 3 (red), 6 (blue), 12 (green), 20 (magenta) and 40 (cyan) min. Time points for ¹³CO data are: 0 (black), 0.67 (red), 1.67 (blue) and 4 (green) min. The ¹³CO signal is enlarged 2.64-fold.

Figure 4

Time courses of thermal recombination at various temperatures. The temperatures at which the recombinations were observed and the fitted exponential rate constants are indicated next to the curves.

Figure 5

Temperature dependence of the recombination rate of CO with A_red* to reform A_red-CO Arrhenius plot based on the data shown in Fig. 4.

Figure 6

EPR spectra of A_red* and A_red-CO (A) 20 mW X-band EPR spectra of A_red-CO in 50 % glycerol (v/v) (black) and A_red-CO after one hour of photolysis at 4.7 K (red line). (Inset) NiFeC signal measured using 320 nW of microwave power (B) Difference of the two spectra in panel A (blue line) and corresponding simulation (green line). See text for spectrometer settings and full simulation parameters.

Figure 7

X-band EPR spectra of reduced, methylated and acetylated ACS preparations. EPR spectra of A_red (top two spectra), A_red–CH₃ (middle), and A_red–COCH₃ (bottom) in 50 % glycerol (v/v) before (dark) and after (“illum”) one hour of white light illumination. In all cases, the spectrum of the buffer was subtracted. Spectrometer settings are identical to those used to acquire the data in Figure 6.

Figure 8

Time course for recovery of NiFeC signal following illumination. EPR spectral intensity of the NiFeC signal in A_red–CO with 50 % glycerol (v/v) as a function of time and white-light illumination (designated by the hash marks). Spectrometer settings: temperature 20.6 K, microwave frequency 9.4744 GHz, microwave power 6.41 μW, modulation frequency 100 kHz, modulation amplitude 0.5 mT, sweep rate 0.73 mT/sec.