Mössbauer Evidence for an Exchange-Coupled {[Fe₄S₄]¹⁺:Ni_p¹⁺} A-cluster in Isolated Alpha Subunits of Acetyl-Coenzyme A Synthase/Carbon Monoxide Dehydrogenase

Abstract

The active site A-cluster in the α subunit of the title enzyme consists of an Fe₄S₄ cluster coordinated to a [Ni_p Ni_d] subcomponent. The cluster must be activated for catalysis using low-potential reductants such as Ti(III) citrate. Relative to the inactive {[Fe₄S₄]²⁺ Ni_p²⁺ Ni_d²⁺} state, the activated state appears to be 2-electrons more reduced, but the location of these electrons within the A-cluster is uncertain, with {[Fe₄S₄]²⁺ Ni_p⁰ Ni_d²⁺} and {[Fe₄S₄]¹⁺ Ni_p¹⁺ Ni_d²⁺} configurations proposed. Recombinant apo-α subunits oligomerize after activation with NiCl₂. The dimer fraction, upon reduction with excess Ti(III)citrate, exhibited Mössbauer spectra consisting of 2 quadrupole doublets representing 51% and 21% of the Fe, with parameters indicating [Fe₄S₄]¹⁺ states. Spectra recorded in strong magnetic fields were typical of diamagnetic systems, indicating an exchange-coupled S = 0 {[Fe₄S₄]¹⁺ Ni_p¹⁺} state. Additional treatment with CO altered the doublet Mössbauer parameters, suggesting an interaction with CO, but maintaining the cluster in the {[Fe₄S₄]¹⁺ Ni_p¹⁺} state. Reduction with sub-stoichiometric equivalents of Ti(III) citrate afforded an EPR signal typical of Ni¹⁺ ions, with g_∥ = 2.10 and g_⊥ = 2.02. Addition of more Ti caused the signal intensity to decline, suggesting that it arises from the semi-reduced {[Fe₄S₄]²⁺ Ni_p¹⁺} state.

Acetyl-coenzyme A synthase/carbon monoxide dehydrogenase is an α₂β₂ tetramer, found in anaerobic archaea and bacteria that grow chemoautotrophically on CO or CO₂.¹ The active site A-cluster in the synthase α subunit consists of an Fe₄S₄ cluster coordinated through a bridging cysteinate to a [Ni_p Ni_d] subcomponent (Chart 1).² The oxidized state of the cluster assembly, A_ox, can be formulated as {[Fe₄S₄]²⁺ Ni_p²⁺ Ni_d²⁺}.^3-5 A_ox does not accept, in the presumed first catalytic step, a methyl cation from the corrinoid-iron-sulfur protein (CH₃-Co³⁺FeSP).⁶ However, methyl transfer occurs when A_ox is treated with low-potential reductants such as Ti(III) citrate. The electronic configuration for the reductively activated A_red-act has not been established but the state is probably 2 electrons more reduced than A_ox.⁴ Reduction of the square-planar distal Ni_d²⁺ with amide N coordination is unlikely,⁷ and stopped-flow evidence disfavors the reduction of the [Fe₄S₄]²⁺ cluster,⁶ leaving the proximal Ni_p²⁺ as the only site that might accept electrons. Attainment of a Nip⁰ state has been suggested,⁵ and this idea is supported by a report that a Ni⁰ phosphine complex can accept a methyl cation from a CH₃-Co³⁺ complex.⁸ However, independent DFT studies by Brunold and Field argue against this suggestion and support a {[Fe₄S₄]¹⁺ Ni_p¹⁺} configuration for A_red-act.^{9, 10} This hypothetical state was not observed in our previous Mössbauer study where it was found that Ti(III) citrate-reduced Ni-activated recombinant α subunits exhibited two major species: ∼ 70 % of spectral intensity originated from {[Fe₄S₄]¹⁺ Ni_p²⁺} centers while ∼ 30 % reflected a diamagnetic state containing an [Fe₄S₄]²⁺ moiety.⁴ Given that the enzyme is known to be heterogeneous, with ∼ 30 % functional and ∼ 70 % nonfunctional A-centers,¹¹ this result was consistent with a {[Fe₄S₄]²⁺ Ni_p⁰} state in functional subunits.

Chart 1.

Structure of the A-cluster. SR represents cysteinyl residues.

We recently altered our procedure for preparing recombinant α subunit by adding NiCl₂ to purified apo-α subunit (containing the Fe₄S₄ cluster but lacking the Ni subcomponent) rather than including it in the growth medium.¹¹ Gel filtration FPLC of Ni-activated α subunits indicated Ni-dependent oligomerization into dimers and other forms.¹² Here we report Mössbauer evidence that α dimer samples prepared in this manner, when reduced by Ti(III) citrate, contain A-clusters in the {[Fe₄S₄]¹⁺ Ni_p¹⁺} state. We also report a new EPR signal that appears to arise from the semi-reduced {[Fe₄S₄]²⁺ Ni_p¹⁺} state.

The 4.2 K Mössbauer spectrum of Ni-activated α dimers reduced with Ti(III) citrate (Figure 1A) exhibits three components. A paramagnetic component (≈ 20-30 % of Fe), extending from −2 mm/s to +2 mm/s Doppler velocity, most probably belongs to {[Fe₄S₄]¹⁺ Ni_p²⁺} clusters. The central portion of the spectrum exhibits two doublets (1 and 2) with ΔE_Q1 = 1.21(4) mm/s, δ₁ = 0.56(2) mm/s, 51(3) % of Fe, and ΔE_Q2 = 0.47(2) mm/s, δ₂ = 0.55(2) mm/s, 21(2) %. The relative intensities of 1 and 2 are ≈ 2.4:1 rather than 2:2 or 3:1 as one might expect if doublets 1 and 2 would represent subsites in one [Fe₄S₄] cluster. The [Fe₄S₄]²⁺ cubane of the A-cluster has δ = 0.45(2) mm/s in all states examined,³ while [Fe₄S₄]¹⁺ clusters typically have an average δ ∼ 0.54-0.59 mm/s.^{13, 14} The S_c = ½ [Fe₄S₄]¹⁺ cluster of apo-α has δ = 0.55 mm/s and ΔE_Q = 0.92 mm/s (Figure S1). Thus, the values for δ1 and δ₂ indicate the [Fe₄S₄]¹⁺ state.

Figure 1.

4.2 K Mössbauer spectra of α dimer taken in applied fields as indicated. (A) Spectrum of 1 mM Ti(III) citrate-reduced α dimer. Red solid line is a superposition of doublets 1 and 2, using the parameters quoted in the text; blue and green solid lines show doublets separately. (B) Spectrum of (A) after removing the line width contribution of the ⁵⁷Co source by a Fourier deconvolution. (C) Spectrum of 1 mM Ti(III) citrate-reduced α dimer after exposure to CO. The solid line is a simulation for doublets 3 and 4 superimposed on a simulation for the A_red-CO species (dashed line above data, using published parameters³). (D) 7.0 T spectrum (parallel applied field) of the sample of (A) after removal of a 25 % contribution of apo-α. The solid line is a spectral simulation assuming that 1 and 2 are diamagnetic; raw data and all parameters are given in Supplementary Information.

Magnetically isolated S_c = ½ [Fe₄S₄]¹⁺ clusters generally exhibit paramagnetic hyperfine structure at 4.2 K (e.g. Figure S1), reflecting hyperfine fields up to ≈ 12 T. The observation of doublets 1 and 2 at 4.2 K suggests A-clusters with integer or zero electronic cluster spin. This argument is supported by spectra recorded in applied fields of 0.1 T, 8 T and 7.0 T (Figure 1D), which show that 1 and 2 belong to diamagnetic A-clusters, strongly suggesting exchange coupling between an S_c = ½ [Fe₄S₄]¹⁺ cluster and an S_Ni = ½ Ni_p¹⁺ site.

Figure 1C shows a 4.2 K spectrum of Ti (III) citrate-reduced α dimers after incubation with CO. This treatment produces the S = ½ NiFeC signal, the signature of the exchange coupled, {[Fe₄S₄]²⁺ Ni_p¹⁺-CO}, A_red-CO state.^{3, 15} In one preparation, we obtained by EPR 0.39(4) spins/α together with the corresponding Mössbauer component, representing 34(3) % of the Fe. For a second preparation (Figure 1C) 31(2) % of the A-clusters were in the A_red-CO state (dashed line drawn above Figure 1C). The remainder of the sample exhibits two doublets, 3 and 4, again reflecting [Fe₄S₄]¹⁺ clusters albeit with parameters that differ from the Ti(III) citrate-reduced sample: ΔE_Q3 = 0.96 mm/s, δ₃ = 0.51 mm/s, 42(3) % of Fe, and ΔE_Q4 = 1.88 mm/s, δ₄ = 0.60 mm/s, 21(2) %. Interestingly, addition of CO changed all spectral components. We speculate that 3 and 4 reflect a {[Fe₄S₄]¹⁺ Ni_p¹⁺-CO} cluster.¹⁶ The data of Figure 1C also imply that all α subunits in the sample have the proximal site occupied by Ni. Studies at 8.0 T applied field (Figure S3) show that 3 (and probably 4) originates from a diamagnetic site.

The fractions quoted for 1,2 and 3,4 do not reveal whether doublets 1,2 (and 3,4) belong to the same cluster; we defer this problem to future studies. Here we discuss aspects of the electronic structure of the A-cluster implied by the observation of diamagnetic species with δ ≈ 0.55-0.60 mm/s, using 1 as an example.

[Fe₄S₄]¹⁺ clusters occur in various spin states, mostly with S_c = ½ and 3/2. We ignore S_c = 3/2 configurations here as they do not yield a diamagnetic ground state for {[Fe₄S₄]¹⁺ Ni_p¹⁺}. Frequently, the S_c = ½ ground state of [Fe₄S₄]¹⁺ clusters yields a diferrous and mixed-valence pair, distinguishable by their ΔE_Q and δ values.¹³ However, some [Fe₄S₄]¹⁺ clusters,¹⁷ like that of apo-α of Figure S1, have the same ΔE_Q and δ for all sites. The exchange-coupling pathway between the S_c = ½ [Fe₄S₄]¹⁺ cluster and the S_Ni = ½ Ni_p¹⁺ involves the cysteinate that links one cluster site, previously labeled Fe_D, to the Ni¹⁺ (ℋ = JS_D•S_Ni, where S_D is the local spin of cluster site D).³ The J-term acts in first-order between the two S = ½ ground manifolds and mixes in second-order excited [Fe₄S₄]¹⁺ cluster states into the ground manifold. The second-order term is relevant for understanding the A_red-CO state³ but we ignore it here. Coupling then yields a singlet (A-cluster spin S = 0) and a triplet state, split by K_DJ, where K_D is a spin projection factor (|K_D| ≤ 1.5 ¹⁸) that depends on the internal coupling of the [Fe₄S₄]¹⁺ cluster and the nature of Fe_D. For K_DJ > 0 the ground state is diamagnetic. The solid line in Figure 1D is a spectral simulation assuming that 1 and 2 are diamagnetic.

In a series of preliminary experiments we observed a new EPR signal when samples of Ni-activated α were treated with sub-stoichiometric amounts of Ti(III) citrate. This axial signal (Figure 2A) has g_∥ = 2.10 and g_⊥ = 2.02, values typical of Ni¹⁺ species. The intensity of the axial signal increases as Ti(III) citrate is initially added (Figure 2B) and maximizes at ∼ 0.2 equivalents/α of Ti(III) citrate (See also Supplementary Information). The qualitative behavior of the titration plot suggests that the signal originates from a semi-reduced S = ½ {[Fe₄S₄]²⁺ Ni_p¹⁺} state which is then further reduced to the {[Fe₄S₄]¹⁺ Ni_p¹⁺} state.

Figure 2.

(A) EPR spectrum of Ni-activated α dimer reduced with 0.2 Ti(III) citrate/α monomer. Conditions: 9.482 GHz; 1 mT modulation; T = 10 K. (B) Plot of EPR signal intensity (amplitude of derivative feature) versus amount of Ti(III) citrate added.

After the Mössbauer spectra were collected, the two samples of Figure 1 were analyzed for their ability to accept a methyl group from CoFeSP. In each case, 0.3 methyl groups per α were transferred, indicating that only ∼ 30 % of the α subunits in these samples arise from the functional A_red-act state.

In summary, our results demonstrate that Ti(III) citrate can reduce the isolated Ni-activated α subunit of the title enzyme to form the {[Fe₄S₄]¹⁺ Ni_p¹⁺} state, and what appears to be the semi-reduced {[Fe₄S₄]²⁺ Ni_p¹⁺} state. The former state has been suggested by DFT calculations, and the latter has been proposed as a catalytic intermediate¹ but here we provide the first experimental evidence for the existence of these states. Although further studies are required to reveal which of the Mössbauer spectral components exhibited by the Ti(III) citrate-treated sample represent the A_red-act state, binding of a methyl cation to {[Fe₄S₄]¹⁺ Ni_p¹⁺} could result, after internal electron transfer from the [Fe₄S₄]¹⁺ moiety to Ni_p, in the methyl-bound state {[Fe₄S₄]²⁺ Ni_p²⁺-CH₃}.⁴