Probing the electronic and mechanistic roles of the μ4-sulfur atom in a synthetic CuZ model system

Nitrous oxide (N2O) contributes significantly to ozone layer depletion and is a potent greenhouse agent, motivating interest in the chemical details of biological N2O fixation by nitrous oxide reductase (N2OR) during bacterial denitrification. In this study, we report a combined experimental/computational study of a synthetic [4Cu:1S] cluster supported by N-donor ligands that can be considered the closest structural and functional mimic of the CuZ catalytic site in N2OR reported to date. Quantitative N2 measurements during synthetic N2O reduction were used to determine reaction stoichiometry, which in turn was used as the basis for density functional theory (DFT) modeling of hypothetical reaction intermediates. The mechanism for N2O reduction emerging from this computational modeling involves cooperative activation of N2O across a Cu/S cluster edge. Direct interaction of the μ4-S ligand with the N2O substrate during coordination and N–O bond cleavage represents an unconventional mechanistic paradigm to be considered for the chemistry of CuZ and related metal–sulfur clusters. Consistent with hypothetical participation of the μ4-S unit in two-electron reduction of N2O, Cu K-edge and S K-edge X-ray absorption spectroscopy (XAS) reveal a high degree of participation by the μ4-S in redox changes, with approximately 21% S 3p contribution to the redox-active molecular orbital in the highly covalent [4Cu:1S] core, compared to approximately 14% Cu 3d contribution per copper. The XAS data included in this study represent the first spectroscopic interrogation of multiple redox levels of a [4Cu:1S] cluster and show high fidelity to the biological CuZ site.


Introduction
Metal-sulde clusters represent a common motif in bioinorganic chemistry. The most studied examples are iron-sulfur clusters (e.g. [2Fe:2S], [4Fe:4S], etc.) that serve as ubiquitous electron transfer sites in a wide range of metalloproteins. 1 Other scenarios such as the [NiFe] and [MoCu] catalytic sites of carbon monoxide dehydrogenases (CODHs), 2,3 the H-cluster found in [FeFe] hydrogenases, and the [FeMo]-cofactor of nitrogenases 4 involve multinuclear metal-sulde clusters facilitating multielectron/multiproton catalytic transformations. Typically, the bridging suldo (S 2À ) ligands in these clusters are thought to be crucial for electronically coupling the transition metal sites, thereby facilitating electron delocalization and lowering barriers towards electron transfer either to/from a catalytic site or along an electron transport chain. However, only in rare cases are the sulfur centers proposed to play a direct rather than spectator role with regard to bond activation and/or bond formation. In the case of the [FeMo]-cofactor of nitrogenase, various hypotheses have been put forward in which sulde ligands might act as redox-active proton relays 5 or even that sulde(s) may serve in a gating mechanism to nitrogenase activity. 6 In the case of the [MoCu] catalytic site of aerobic CODH, one mechanistic hypothesis based on crystallographic studies with substrate analogues involves the m 2 -sulde ligand actively participating in CO activation via transient S-C bond formation. 2 During bacterial denitrication, nitrous oxide (N 2 O) is converted to N 2 + H 2 O in a 2e À /2H + reaction catalyzed by the metalloenzyme, nitrous oxide reductase (N 2 OR). 7 The catalytic site of N 2 OR is a tetranuclear copper-sulfur cluster, Cu Z , which has been structurally characterized in both [4Cu:1S] and [4Cu:2S] forms. 8,9 Both forms show N 2 O reductase activity to some extent, and both require physiological reduction to their most reduced redox states to activate N 2 O: the 4Cu I ("fully reduced") state for the [4Cu:1S] cluster and the 3Cu I :1Cu II ("1-hole") state for the [4Cu:2S] cluster. 10 For the [4Cu:1S] cluster, Solomon has proposed N 2 O binding across a dicopper(I) cluster edge, with the N 2 O molecule occupying a m-1,3 binding mode, based on computational modeling (Fig. 1a). 11 For the [4Cu:2S] form, Einsle has reported crystallographic data on N 2 O-pressurized crystals of N 2 OR showing a N 2 O molecule within van der Waals contact of Cu Z , but the N 2 O molecule was not found within coordination distance of Cu Z and had not undergone signicant activation (Fig. 1b). 9 In neither case has experimental data emerged to probe the nature of N 2 O activation by the copper-sulfur clusters.
Studying synthetic model systems can aid understanding of how these unusual inorganic copper-sulfur functional groups behave, 12 which is particularly crucial knowledge in the context of N 2 O's signicant impact as a greenhouse gas and an ozone layer depleting agent. 13,14 Among the synthetic copper compounds and materials known to activate N 2 O, 15-18 one of our groups has reported the only examples of N 2 O activation by copper sulde clusters. In one case, a dicuprous [Cu 2 S] cluster with an unsupported m 2 -sulde bridge 19 was found to reduce multiple N 2 O equivalents to N 2 , resulting in exhaustive oxidation of the sulfur center to a m 2 -sulfate ligand (Scheme 1a). 20 Here, the copper centers remained redox inactive while the m 2 -sulde ligand was not only the redox-active center but also acted as an oxygen atom acceptor. In another case, a phosphine-supported tetranuclear [Cu 4 S] cluster in its 4Cu I state showed reactivity towards N 2 O reduction, 21 but the cluster lost structural integrity during the reaction, losing the sulfur atom to unknown products in the reaction medium and thus limiting insight that can be gained about its role. Finally, a formamidinate-supported [Cu 4 S] cluster in its formally 3Cu I :1Cu II ([4Cu:1S] 1À ) state was found to reduce 15 N 2 O to 15 N 2 (Scheme 1b). 22,23 Here the m 4 -sulde bridge remained intact during a formal oxidation to the 2Cu I :2Cu II ([4Cu:1S] 0 ) redox state of the cluster, allowing us to establish a closed cycle for N 2 O reduction. Based on these results, the potential role (or lack thereof) of the bridging sulde ligand in copper-sulfur clusters merits further investigation.
In this report, we disclose a combined experimental/ computational study of the latter system that collectively implicates the m 4 -sulde ligand as participating in redox changes and directly interacting with N 2 O during its activation (Scheme 1c). Our data includes the rst spectroscopic interrogation of multiple [4Cu:1S] redox levels, which has proven challenging in the metalloenzyme system, 7,24 and highlights the delity of our synthetic model to the biological Cu Z site. Additionally, the direct interaction of N 2 O with the bridging sulfur atom(s) in Cu Z has not been proposed before. Such reaction pathways should be considered for the chemistry of Cu Z and related metal-sulde clusters in light of the synthetic model studies reported herein.

Results and discussion
In our previous report of N 2 4 ] (the 2-hole cluster, referred to here as [4Cu:1S] 0 ), N 2 , and O 2À . However, we were unable to denitively establish the reaction stoichiometry at that time. Since then, we have undertaken quantitative GC-MS analysis of the reaction headspace to determine the yield of N 2 . According to this analysis (see ESI †), 0.53 AE 0.06 mol of N 2 are produced per mol of the [4Cu:1S] 1À complex. When combining this result with our previous observations, we can now condently propose the balanced reaction shown in Scheme 2 as the dominant pathway. Based on this reaction stoichiometry, we proceeded with the working hypothesis that one equivalent of [4Cu:1S] 1À is responsible for N 2 O activation while a second equivalent is acting as a sacricial reductant, thus accounting for the overall two-electron redox reaction. Next, because we have been unable to detect any intermediates experimentally, we sought to examine the binding mode of N 2 O using DFT modeling at the B3LYP/6-31G(d) level in the gas phase. To save computational time, the mesityl groups on the supporting NCN ligands were replaced with methyl groups.  2À ). In both cases N 2 O occupied a m-1,3 binding mode, but to our surprise the N 2 O molecule was found to bridge one of the Cu centers and the S atom (Fig. 2a). In each case, one of the other Cu centers has moved away from the S atom to facilitate its direct interaction with N 2 O. An alternative, m 3 -1,2 binding mode in which the N 2 O molecule bridges two Cu centers as well as the S atom also was located but was determined to be signicantly higher in energy by +11.8 kcal mol À1 on the Gibbs free energy surface (see ESI †). The preferred binding mode for this model system is distinct from the m-1,3 bridging between two Cu centers that is proposed for Cu Z (see Fig. 1a), where the m 4 -S 2À ligand is not proposed to interact directly with N 2 O. It should be noted that a mononuclear intermediate in which N 2 O bridges across a terminal nickel-sulde bond has been isolated and crystallographically characterized by Hayton and coworkers. 25,26 The accord between the metrical parameters of the activated N 2 O in our computational model with Hayton's experimental data (Fig. 2b)  Because the m 4 -sulde ligand seems to play a crucial and direct role in N 2 O activation according to our DFT modeling, we wondered whether the frontier orbitals of these synthetic [4Cu:1S] complexes have notable sulfur character. In order to validate our mechanistic model, we thus undertook multi-edge X-ray absorption spectroscopy (XAS) combined with higher-level computational modeling to interrogate the electronic structural changes underpinning the [4Cu:1S] 0/1À redox process.
Cu K-edge XAS data obtained for [4Cu:1S] 1À and [4Cu:1S] 0 are shown in Fig. 3a. Spectral subtraction was carried out to remove a minor contribution of [4Cu:1S] 0 in the spectrum of the monoanion (vide infra). Neither spectrum presents a resolved pre-edge (1s / 3d) feature, although both spectra feature a shoulder that gives a peak in the second derivative spectrum at 8979.8 eV, consistent with the presence of Cu 3d vacancies (Fig. 3b). The rising edges of the two spectra have qualitatively similar ne structure including maxima at ca. 8983 eV suggesting the presence of Cu I centers, 28 although the spectrum of the [4Cu:1S] 1À cluster is shied, with inection points occurring at 0.8 to 1.1 eV lower energy relative to [4Cu:1S] 0 . Given the effectively identical coordination environments between the two species, the shi in rising edge position largely reects some Cu participation in the redox process. Moreover, the lack Quantitative estimates of S participation in the redox-active molecular orbital (RAMO) can be gleaned through analysis of S K-edge XAS data 29 obtained for the two clusters, which are Scheme 3 Reaction pathways modeled by DFT (B3LYP/6-31G(d)). Energies at 298 K are shown in kcal mol À1 . The favored pathway is shown with solid arrows, and the disfavored pathway with dotted arrows. presented in Fig. 4. Well-resolved pre-edge peaks are apparent in both spectra, occurring at 2470.  (Fig. S15 †). Notably, the 2469.5 eV [4Cu:1S] 1À pre-edge peak energy value closely matches pre-edge peak energies reported by Solomon and co-workers for the Cu Z sites of resting Achromobacter cycloclastes 31 and Paracoccus denitricans 30 N 2 OR at 2469.2 and 2469.0 eV, respectively. On the basis of Cu K-edge XAS analysis, Solomon and co-workers assigned resting Cu Z as a 3Cu I :1Cu II cluster, 30 consistent with the formal oxidation state distribution expected for the [4Cu:1S] 1À cluster. Pre-edge peaks in the S K-edge XAS spectra of metal complexes and clusters bearing S-donor ligands reect excitations from S 1s / c*, where c*, the acceptor MO, is an antibonding ligand eld MO born of metal-sulfur mixing: where a 2 reects the % 3p contribution in the acceptor MO. 29 Pre-edge peak intensities (D 0 ) are then given by the relationship: where h is the number of holes in the acceptor MO, n is the number of photoabsorbing nuclei from which electrons can be excited into the acceptor MO, and I s is the radial dipole integral h3p|r|1si governing the intensity of a "pure" S 1s / 3p excitation. Solomon and co-workers 32 have estimated the value of I s as a function of the S 1s / 4p excitation energy, which can itself be gleaned from S K-edge XAS data and will vary according to the nature of the S photoabsorber and its chemical environment. Using TDDFT calculations to facilitate the assignments  Fig. 5. These are qualitatively similar, indicating that the RAMO is a highly delocalized orbital featuring effectively equal participation of Cu 3d from all 4 metal centers along with a signicant contribution from S 3p. Equal participation of all three Cu centers in the SOMO was previously indicated by simulation of experimental EPR parameters. 22 The equal contributions from Cu are also in accord with observation that the Cu K-edge XANES shi in energy but do not exhibit differences in ne structure. Calculated S 3p contributions are 20.6% for [4Cu:1S] 0 and 21.1% for [4Cu:1S] 1À , in splendid agreement with experiment as well as with previous EPR analysis of the [4Cu:1S] 1À species that indicated anomalously small Cu hyperne coupling. 22 Moreover, TDDFT calculations 37 of the S K-edge XAS for both species initiated from the aforementioned single-point DFT calculations give spectra that nicely reproduce  the energy and intensity differences encountered in the experimental data (Fig. 6).

Conclusions
We previously reported that the 1-hole [4Cu:1S] 1À model cluster is oxidized to its 2-hole state by N 2 O with N 2 evolution. 22 Here, we have measured the reaction stoichiometry, allowing us to conclude that the overall 2-electron reduction of N 2 O requires two equivalents of the [4Cu:1S] 1À cluster molecule, with each equivalent mediating a 1-electron redox process individually. Under the assumption that one equivalent activates N 2 O while the other acts as a sacricial reductant, a computational model of the reaction intermediates indicated cooperative Cu/S coordination of N 2 O.
This cooperative binding mode implies direct participation of the bridging S-atom in N 2 O activation and N-O cleavage, in contrast to the passive role of bridging S-atoms in typical metalsulfur active sites. Consistent with this proposal, XAS analysis of the 1-hole and 2-hole clusters indicated that the m 4 -S center contributes appreciably to the redox-active molecular orbital. Crucially, the S K-edge energies and estimated S-atom participation in redox chemistry closely match previous characterization of the biological Cu Z site, making this synthetic system a faithful model in terms of electronic structure as well as atomic connectivity and chemical reactivity. Moreover, to our knowledge this data represents the rst spectroscopic interrogation of multiple redox levels of a conserved [4Cu:1S] cluster. Key to the model cluster's reactivity, and in particular to the m 4 -S center's active participation in N 2 O activation and reduction, is the high degree of covalency within the [4Cu:1S] core. This Cu/S covalency allows the S-atom to exhibit characteristics typically associated with transition metals, such as the ability to simultaneously accept and donate electron density to/from the substrate and to vary its oxidation level during a chemical process, that are necessary for a catalytic active site mediating a multielectron redox process. Thus, it is important to consider both metal/metal and metal/ligand cooperation when interrogating highly covalent multinuclear catalysts such as Cu Z and related systems.

Conflicts of interest
There are no conicts to declare.