Evidence that ferredoxin interfaces with an internal redox shuttle in Acetyl-CoA synthase during reductive activation and catalysis

Abstract

Acetyl-CoA synthase (ACS), a subunit of the bifunctional CO dehydrogenase/acetyl-CoA synthase (CODH/ACS) complex of Moorella thermoacetica requires reductive activation in order to catalyze acetyl-CoA synthesis and related partial reactions, including the CO/[1-(14)C]-acetyl-CoA exchange reaction. We show that the M. thermoacetica ferredoxin(II) (Fd-II), which harbors two [4Fe-4S] clusters and is an electron acceptor for CODH, serves as a redox activator of ACS. The level of activation depends on the oxidation states of both ACS and Fd-II, which strongly suggests that Fd-II acts as a reducing agent. By the use of controlled potential enzymology, the midpoint reduction potential for the catalytic one-electron redox-active species in the CO/acetyl-CoA exchange reaction is -511 mV, which is similar to the midpoint reduction potential that was earlier measured for other reactions involving ACS. Incubation of ACS with Fd-II and CO leads to the formation of the NiFeC species, which also supports the role of Fd-II as a reductant for ACS. In addition to being a reductant, Fd-II can accept electrons from acetylated ACS, as observed by the increased intensity of the EPR spectrum of reduced Fd-II, indicating that there is a stored electron within an "electron shuttle" in the acetyl-Ni(II) form of ACS. This "shuttle" is proposed to serve as a redox mediator during activation and at different steps of the ACS catalytic cycle.

Figure 1. UV-visible, EPR and electrochemical characterization of Fd-II

(a) UV-visible absorbance spectra of 11.2 µM Fd-II in 50 mM TRIS (pH=7.6) when it is reduced (solid line) versus oxidized (dashed line). (b) EPR spectra of Fd-II samples that were poised at representative potentials of −550 mV and −440 mV. The EPR parameters were as follows: Frequency, 9.38 GHz; microwave power, 20 mW; receiver gain, 2000; modulation frequency, 100 KHz; and modulation amplitude, 10.00 Gauss. (c) Determination of the midpoint potential for Fd-II. The peak intensity vs. potential plots for peaks at half-field (Δm_s=2, g=3.88, closed circles (●)) and at g-value of 1.934 (▲), which were used to determine fraction of each cluster at the reduced state. The fits were done using the equations described in Methods, and the redox potentials of −454 mV and −487 mV were determined for the two clusters. The half-field transition of the sample poised at −528 mV is shown in the inset.

Figure 2. Reductive activation of ACS by Fd-II

(a) The increase in CO/Acetyl-CoA exchange activity is shown as a function of Fd-II concentration. The assay was done at 55 °C with 4 µM ACS (30% NiFeC) in 0.3 M MES (pH=6.2), 200 µM acetyl-CoA, 5 µM ¹⁴C-labeled acetyl-CoA, ~700 µM CO and titrated with different concentrations of Fd-II between 0 and 150 µM. The activation curve was fit with a Hill coefficient of 2.2 ± 0.7, and the apparent dissociation constant was estimated to be 36.9 ± 6.3 µM. (b) The reduction of ACS by Fd-II was monitored by measuring the formation of the NiFeC signal. The molar equivalencies of Fd-II were 0, 0.5 and 3. The EPR parameters were the same as in figure 1 except that the microwave power here was 1 mW.

Figure 3. The effect of modulation of the redox states of Fd-II and ACS on ACS activity

The radioactive acetyl-CoA decay curves from CO/acetyl-CoA exchange assays at 55 °C using either unactivated ACS (a) or ACS prereduced by 10 molar equivalents of dithionite (b). The different oxidation states of Fd-II are symbolized by: x, no Fd-II; ▲, partially oxidized Fd-II; ♦, oxidized Fd-II; and ●, reduced Fd-II. The concentration of ACS (50% NiFeC) was 4 µM in (a) and 1.8 µM in (b). The concentration of Fd-II was 30 µM in all reactions. All the concentrations of other ingredients were the same as on Figure 2 legend. The specific activities obtained from the reactions are summarized in Table 1.

Figure 4. Dependence of ACS activity on solution redox potential

The specific activity of the CO/Acetyl-CoA exchange reaction was measured at different solution potentials at 27 °C in an electrochemical cell in the anaerobic chamber. The buffer and the concentrations of different ingredients were the same as in Figure 2 legend, except for ACS, which was 20 µM. In addition, 100 µM each of the following redox mediators was included: 4,4'-dimethyl,2,2'-dipyridyl, methyl viologen, benzyl viologen and Triquat. The trend in specific activity was plotted using either the one-electron (solid line) or the two-electron (dashed line) Nernst equation. The R² value for the one-electron fit is 0.9437, whereas for the two electron fit, it is 0.8583. The midpoint redox potential according to the one-electron Nernst equation is (−511 ± 6) mV.

Figure 5. Measurement of electron transfer from acetylated-ACS to Fd-II

The acetylated ACS sample was reacted with CoA in the presence of oxidized Fd-II (b). Also shown are the EPR spectra of acetylated ACS (a), acetylated ACS incubated with Fd-II without any CoA (c) and oxidized Fd-II by itself (d). All of the reactions were performed in 50 mM KPi buffer (pH 7.5) and 20 mM KCl. The ACS, Fd-II and CoA concentrations were 69 µM, 100 µM and 500 µM respectively. The EPR spectra were taken at 10K, and the parameters were as follows: Frequency, 9.38 GHz; microwave power, 10 µW; receiver gain, 50000; modulation frequency, 100 KHz; modulation amplitude, 10 Gauss.

Scheme 1. Proposed paramagnetic mechanism of ACS with possible Fd-II involvement

This scheme depicts the random nature of the methylation and carbonylation steps of the ACS mechanism, with the “carbonylation-first” mechanism shown above in red and the “methylation-first” mechanism shown below in blue. The scheme also explicitly shows the internal electron shuttle (IES) that equilibrates electrons during reductive activation and at one stage of the catalytic cycle. The numbered block arrows depict the two stages of the ACS reaction studied in this paper. The lower panel shows the interaction of Fd-II with that IES.