A 1-hole Cu₄S cluster with N₂O reductase activity: a structural and functional model for Cu_Z*

Abstract

During bacterial denitrification, two-electron reduction of N₂O occurs at a [Cu₄(μ₄-S)] catalytic site (Cu_Z*) embedded within the nitrous oxide reductase (N₂OR) enzyme. In this communication, an amidinate-supported [Cu₄(μ₄-S)] model cluster in its 1-hole (S = ½) redox state is thoroughly characterized. Along with its 2-hole redox partner and fully reduced clusters reported previously, the new species completes the two-electron redox series of [Cu₄(μ₄-S)] model complexes with catalytically relevant oxidation states for the first time. More importantly, N₂O is reduced by the 1-hole cluster to produce N₂ and the 2-hole cluster, thereby completing a closed cycle for N₂O reduction. The title complex is thus not only the best structural model for Cu_Z* to date, but it also serves as a functional Cu_Z* mimic.

Graphical abstract

Regulation of nitrous oxide (N₂O) concentration in the atmosphere is crucial due to N₂O's key roles both as an anthropogenic greenhouse gas and as an ozone layer depletion agent.^1,2 Lessons can be taken from nature, where atmospheric N₂O concentrations are regulated by the bacterial denitrification metalloenzyme, nitrous oxide reductase (N₂OR).³ The catalytic site in N₂OR that is reactive under biological conditions is Cu_Z*,⁴ a [Cu₄(μ₄-S)] cluster characterized in the resting “1-hole” (3Cu^I:1Cu^II, S = ½) state^5,6 and active in the “fully reduced” (4Cu^I, S = 0) state (Figure 1a).⁷ Under certain conditions, the Cu_Z* site in N₂OR is replaced by Cu_Z,⁸ a [Cu₄(μ₄-S)(μ₂-S)] cluster with a “2-hole” (2Cu^I:2Cu^II, S = 0) resting state that converts to a [Cu₄(μ₄-S)(μ₂-SH)] cluster upon reduction to the 1-hole state, which shows relevant though limited N₂O reductase activity (Figure 1b).⁹ Because N₂OR catalyzes the two-electron reduction of N₂O, three Cu_Z* redox states (4Cu^I, 3Cu^I:1Cu^II, and 2Cu^I:2Cu^II) spanning a two-electron range are plausibly relevant to catalysis.^10,11

Figure 1.

Structures of (a) Cu_Z* (with N₂O bound) and (b) Cu_Z sites of N₂OR; (c) [Cu₄(μ₄-S)] model complexes.

The unique [Cu₄(μ₄-S)] structural motif and the rich redox chemistry of this catalytic site have presented challenges to synthetic modeling chemistry. Synthetic examples of [Cu₄(μ₄-S)] clusters supported by phosphorus ligands have only been isolated in the 4Cu^I state and do not react with N₂O.^12,13 Other relevant models that do access open-shell oxidation states feature [Cu₃(μ₃-S₂)] or [Cu₃(μ₃-S)] cores that do not structurally model Cu_Z*.^14,15 Similarly, functional models capable of N₂O reduction feature [Cu₃(μ₂-S₂)] or [Cu₂(μ₂-SR)] cores,^16,17 limiting mechanistic insight to be gained for comparison to the tetracopper core of Cu_Z*.

We recently reported a [Cu₄(μ₄-S)] cluster (1), supported by nitrogenous amidinate ligands,¹⁸ that was characterized in its 2-hole state. Here, we report the synthesis and characterization of its one-electron reduction product, the 1-hole derivative (2). Along with the fully reduced clusters supported by diphosphine¹² (3a) and diphosphinous amide¹³ (3b) ligands, this completes the catalytically relevant two-electron redox series of [Cu₄(μ₄-S)] model complexes for the first time (Figure 1c). Species 2 reduces N₂O stoichiometrically, producing 1 + N₂ and completing a synthetic cycle for N₂O reduction (Scheme 1). The 1/2 redox pair thus represents both a structural and functional Cu_Z* model system.

Scheme 1. Synthetic cycle for N₂O reduction.

We previously showed that cluster 1 assembles upon addition of S-atom donors to a dicopper(I) bis(amidinate) precursor.¹⁸ The 2-hole, formally 2Cu^I:2Cu^II complex 1 was originally assigned as having a S = 0 ground state and a low-lying S = 1 excited state, the latter based on detection of a temperature-dependent solution magnetic moment and an EPR signal with non-Curie behavior. However, analysis of rigorously purified samples of 1 by SQUID magnetometry reveal near-zero χ_Т values up to 400 K (Figures S1), consistent with a diamagnetic species. Furthermore, one of the side products formed during assembly of 1 was characterized by X-ray crystallography. This monocopper(II) species resulting from S-atom insertion into two Cu-N bonds (Figure S2) gives an EPR signal matching that previously reported for 1 (Figure S3). Rigorously purified samples of 1, on the other hand, are EPR-silent. Considering this new data, we now assign 1 as being diamagnetic, while data consistent with paramagnetism in previous samples are now assigned to trace impurities.

Complex 1 possesses a reversible one-electron redox event at E°' = -1.28 V vs. Fc⁺/Fc (Fc = ferrocene).¹⁸ Chemical reduction of 1 with [K(18-crown-6)₂][Fp] (Fp = FeCp(CO)₂, E°' = -1.8 V vs. Fc⁺/Fc)¹⁹ produced 2 as its [K(18-crown-6)]⁺ salt, along with 1 equiv. of free 18-crown-6 and 0.5 equiv. of Fp₂ (Scheme 1a). X-ray crystallographic analysis of 2 revealed two symmetrically independent tetracopper anions, one of which is shown in Figure 2. Both anions feature close contacts between an amidinate mesityl ring and the nearby [K(18-crown-6)]⁺ unit. Anionic 2 is isostructural to 1 and to dicationic 3a and 3b, with local C_2v symmetry and an alternating up-down-up-down pattern for the bridging amidinates.

Figure 2.

Solid-state structure of anionic 2 as a [K(18-crown-6)]⁺ salt. Hydrogen atoms, co-crystallized solvent, and a symmetrically independent second molecule have been omitted for clarity.

Key structural parameters for the pyramidal [Cu₄(μ₄-S)] pentahedra within 1, 2, and 3a are compared in Table 1. The 2-hole species 1 features a rectangular Cu₄ base, with alternating short and long Cu-Cu distances. Upon reduction to 1-hole 2, the Cu₄ base is less unsymmetric and approaches a square shape, with a smaller difference between short and long Cu-Cu distances. The core of fully reduced 3a is even closer to a square-based pyramid shape. Evidently, there is a well behaved pattern across the redox series: the Cu₄ base gets more rectangular with increasing oxidation level, and gets more square with decreasing oxidation level. The geometry of the four-coordinate S center is less well behaved as a function of redox state, as measured by the τ₄ parameter²⁰ that does not follow a clear pattern across the series. The [Cu₄(μ₄-S)] core of 1-hole Cu_Z* has a seesaw shape rather than a pyramidal shape, with nearest-neighbor Cu-Cu distances spanning 2.56 to 3.36 Å.²¹

Table 1. Redox-dependent [Cu₄(μ₄-S)] bond metrics^a.

	1 (2-hole)b	2 (1-hole)c	3a (0-hole)d
Cu1-Cu2	2.4226(6)	2.502(1)	2.869(1)
Cu2-Cu3	3.0353(6)	2.809(1)	3.128(1)
Cu3-Cu4	2.4226(6)	2.532(1)	2.869(1)
Cu1-Cu4	3.0353(6)	2.831(1)	3.128(1)
τ4(S)e	0.76	0.90	0.59

^aBond distances in Å.

^bFrom ref 18.

^cFor one of two molecules in the asymmetric unit.

^dFrom ref 12.

^eFor μ₄-S ligand: τ₄ is 1.00 for T_d and 0.00 for D_4h, see ref 20.

The S = ½ species 2 was characterized by X-band and Q-band EPR spectroscopy. The g-values for the axial signal were not readily obtained from the X-band spectrum (Figure 3a) but were well resolved in the Q-band spectrum (Figure S4): g_┴ = 2.090 and g_‖ = 2.043. Resolved lines on the high- and low-field sides of the X-band spectrum (Figure S5) were attributed to Cu hyperfine splitting, and values of A_┴ = 100 MHz and A_‖ = 15 MHz were obtained by fitting the X-band and Q-band spectra. The 2^nd derivative X-band spectrum emphasizes fine structure for the 13-line pattern resulting from four equivalent Cu centers, and the simulated spectrum fits the experimental data well (Figure 3b). The Cu hyperfine coupling in 2 is small in magnitude relative to typical cupric species. A previous 1-hole [Cu₃(μ₃-S)] model exhibited an isotropic signal (g = 2.095) with a similarly small Cu hyperfine constant (97 MHz).¹⁵ The EPR signatures for 1-hole Cu_Z* and Cu_Z are distinct from 2 in that they have g_‖ > g_┴ and larger hyperfine constants (Table 2).⁹

Figure 3.

X-band EPR data (9.632 GHz, 9.9 K, 2-MeTHF) for 2 shown as (a) 1^st derivative, (b) 2^nd derivative overlay of simulation (red) and experimental (black); (c) Mulliken spin density plot (0.001 isovalue) for 2′ calculated by DFT.

Table 2. Redox-dependent spectroscopic properties.

	1a	2	CuZb,c	CuZb,d	CuZ*b,d
g‖	–	2.043	–	2.152	2.160
g┴	–	2.090	–	2.042	2.043
A‖e	–	15	–	168	182, 69
A┴e	–	100	–	60	75, 60
λmaxf	561(470)h	566	546(670)h	694	680
εg	14,000i	8,600	10,000i	3,000	4,500

^aFrom ref 18.

^bFrom ref 9.

^c2-hole.

^d1-hole.

^eIn MHz.

^fIn nm.

^gIn M^-1cm^-1.

^hShoulder.

ⁱFor main peak.

Based on the EPR data for 2, the formally 3Cu^I:1Cu^II:S^2- complex can be viewed as an admixture of two limiting resonance contributors: a delocalized 4Cu^1.25:S^2- mixed-valent species, and a 4Cu^I:S^- sulfur-radical species. To our knowledge, the literature of sulfur EPR spectroscopy does not include any four-coordinate examples for comparison to the S center in 2.^22-24 To probe electronic structure further, we analyzed a model complex 2′, in which the mesityl groups had been replaced with methyl groups, using DFT computations. The computed bond distances within the [Cu₄(μ₄-S)] core for 2′ matched experimental values well (Table S1). The Mulliken spin density for 2′ was found to be delocalized, with equal populations on each of the four Cu centers and with the S center having the most spin density (32%) of any single atom (Figure 3c). This computational observation indicates a high degree of covalency in the [Cu₄(μ₄-S)] core.

Complexes 1 and 2 are purple. Complex 1 features a strong absorbance at 561 nm (ε = 14,000 M^-1cm^-1) with a shoulder at 470 nm.¹⁸ Upon reduction (Figure 4a), this feature shifted slightly in 2 to 566 nm and got measurably less intense (ε = 8,600 M^-1cm^-1). Absorption data for Cu_Z* is available only for the 1-hole state. Cu_Z has been characterized in both its 2-hole and 1-hole states (Table 2): a large red-shift and a decrease in intensity are observed upon reduction,⁹ and these transitions previously have been attributed to S^2--to-Cu charge transfer.

Figure 4.

(a) UV-Vis data for 1 (red) and 2 (black); (b) natural transition orbitals (0.04 isovalues) for 578-nm excitement of 2′ calculated by TD-DFT. Relative contributions to NTO 125β: S, 23%; Cu, 14% each.

TD-DFT calculations for 2′ predicted a characteristic feature at 578 nm (ε = 6,000 M^-1cm^-1), and natural transition orbital (NTO) analysis²⁵ indicated that this transition involves excitation of a β-electron from NTO 116β to NTO 125β (Figure 4b). NTO 116β is predominantly a linear combination of four Cu 3d_xz orbitals, while NTO 125β (the LUMO) has significant S 3p_x character. The dominant electronic transition thus clearly involves charge transfer from the four Cu centers to the S center and resembles a delocalized Cu 3d to Cu-S σ* transition. TD-DFT calculations for 1′ correctly predicted an increase in intensity (to ε = 16,000 M^-1cm^-1) and the presence of a shoulder, though not the lack of energy shift.

A reaction was observed when solutions of 2 were exposed to N₂O (1 atm) at -78°C. ¹H NMR analysis indicated that 2 had been oxidized to 1 in up to 89% yield (Scheme 1b). Under certain conditions, evolution of N₂ was detected by headspace GC-MS analysis and comparison to control reactions in the absence of 2 under identical experimental conditions. Evolution of ¹⁵N₂ was detected when ¹⁵N₂O was used, verifying that the liberated nitrogen derived from nitrous oxide. Addition of electrophiles Me₃SiCl or PhC(O)Cl to the final product mixtures produced (Me₃Si)₂O or PhC(O)OC(O)Ph, consistent with the presence of nucleophilic O^2-. Collectively, these observations establish that the reaction shown in Equation 1 was taking place. Due to difficulties in accurately quantifying the N₂ and O^2- produced, the value of n in Equation 1 is ambiguous at this time. Our working hypothesis is that two molecules of 2 cooperate to reduce N₂O by two electrons, with one cluster activating the N₂O substrate and the other acting as a sacrificial reductant. Regardless, complex 2 is the first synthetic [Cu₄S] complex to exhibit N₂O reactivity, and thus opens a new avenue of investigation in N₂O reductase research. Ongoing studies in our laboratory are aimed at detecting intermediates along the N₂O reduction pathway and elucidating the reduction mechanism.

$$2+n{\mathrm{N}}_2\mathrm{O}\to 1+n{\mathrm{N}}_2+n{\mathrm{O}}^{2-}$$ (Eq. 1)

In conclusion, the first 1-hole [Cu₄(μ₄-S)] complex has been synthesized and thoroughly characterized, completing the two-electron redox series of [Cu₄(μ₄-S)] model complexes. Structural, spectroscopic, and computational evidence is consistent with highly covalent bonding within the [Cu₄(μ₄-S)] core. This redox-active [Cu₄(μ₄-S)] system is also a functional mimic for Cu_Z*, participating in a synthetic cycle for N₂O reduction. The title compound thus can be viewed as both a structural and functional model for Cu_Z*.